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Molecular characteristics of permanganate- and dichromateoxidation-resistant soil organic matter from a black-C-rich colluvial soil.

Introduction

Soil organic matter (SOM) is a complex mixture of plant, animal and microbial tissue, both fresh and at different stages of decomposition (Stevenson 1994; Tabatabai 1996). The molecular structures present in SOM have been a source of debate for many decades due to the analytical difficulties inherent to SOM characterisation (Piccolo 1996). Diverse methodologies, both fractionation and analytical, have been used to study SOM composition. Fractionation methods include (z) chemolytic techniques (i.e. application of acid-hydrolysis or extracting agents) that are coupled with colourimetric and/or GC/MS analyses to identify specific SOM components (polysaccharides, lignin-derived compounds, amino sugars, extractable lipids or hydrolysablc proteins) (Kogel-Knabner 1995); (ii) physical fractionation into organo-mineral fractions based on particle size and/or density yields (Christensen 1992; Six et al. 2002); and (iii) wet oxidation with potassium permanganate (KMn[O.sub.4]) (Loginow et al. 1987; Tirol-Padrc and Ladha 2004), [H.sub.2][O.sub.2] (Eusterhues et al. 2005), [Na.sub.2][S.sub.2][O.sub.8] (Eusterhues et al. 2003), NaOCl (Kleber et al. 2005), and [K.sub.2][Cr.sub.2][O.sub.7] (Skjemstad and Taylor 1999). Analytical techniques include (z) spectroscopic techniques, such as infrared (IR) spectroscopy and solid-state [sup.13]C nuclear magnetic resonance ([sup.13]C NMR) (Wilson et al. 1981; Frund et al. 1994) and (it) pyrolysis-GC/MS (Py-GC/MS) (Saiz-Jimenez and de Leeuw 1986). Whereas IR and [sup.13]C NMR provide information on the environment of carbon atoms (functional groups) (Baldock and Smemik 2002), more detail on the molecular chemistry is obtained by pyrolysis-GC/MS.

The Py-GC/MS technique is based on thermal degradation in an inert atmosphere (pyrolysis) and subsequent separation (GC) and identification (MS) of the pyrolyzate, from which information on macromolecular structures can be extracted (Moldoveanu 1998). For example, it allows the identification of different sources and degradation states of plant detritus, secondary/microbial material, black C (BC), and the estimation of their relative proportions (Nierop et al. 2005; Buurman et al. 2007). Nonetheless, Py-GC/MS is a semi-quantitative method because there are differences in the pyrolyzability of different organic matter components, not all pyrolysis products are amenable and detectable by GC, and there are differences in relative response factors by MS (Saiz-Jimenez 1994u).

The Walkley-Black dichromate oxidation, as modified by Heanes (1984), is a relatively simple and rapid procedure with minimal equipment needs (Nelson and Sommers 1996) that has long been used to estimate the organic C (OC) content of soils. Its major disadvantage is that it incompletely oxidises soil OC (Giliman et al. 1986) and has different oxidation efficiencies for different soils (Tabatabai 1996), which produce considerable and unpredictable deviations from 'true' soil OC content. Probable causes of deviations in recoveries are (i) spatial inaccessibility of organic substrates to the oxidation agent (Skjemstad et al. 1996; Six et al. 2002), (ii) binding with inorganic phases (Eusterhues et al. 2005), and (iii) the presence of chemically recalcitrant SOM fractions such as BC (Six et al. 2002). In fact, the difference between total OC and dichromate-oxidisable OC has been used to estimate the BC content of soils. The BC is defined as the product resulting from incomplete thermal combustion of vegetation and/or fossil fuels, and is relatively resistant to decomposition (Schmidt et al. 2001). However, an unknown portion of BC is actually oxidised, while some non-pyrogenic SOM may survive the oxidation, e.g. non-hydrolysable aliphatic compounds that resist aqueous dichromate oxidation (Knicker et al. 2007). This implies that the properties of the oxidation-resistant residue must be assessed in order to obtain meaningful estimations of BC content using dichromate oxidation (Knickcr et al. 2007). The stability of BC towards these reagents is of significant interest considering its upcoming use as a soil amendment (as biochar).

The permanganate-oxidisablc fraction has been used as a proxy for the labile fraction of SOM (Loginow et al. 1987; Lefroy et al. 1993), based on the assumption that the oxidative capacity of KMn[O.sub.4] on SOM is comparable to that of soil microbial enzymes (Contch et al. 1997). However, some studies indicated that KMn[O.sub.4]-ox idisable C may not be a reliable measure of the proportion of labile C because, even though it efficiently degrades lignin (van Soest and Wine 1986), it has little effect on several SOM components that are widely recognised as easily degraded by soil microorganisms, e.g. structural carbohydrates, sugars and amino acids (e.g. Tirol-Padre and Ladha 2004). Furthermore, its ability to react with charcoal was also stated (Skjemstad et al. 2006).

Soil organic matter is thermodynamically unstable in wellaerated soils (Macias and Camps-Arbestain 2010). However, SOM stabilised by specific mechanisms can remain as metastable forms in soils for hundreds to thousands of years (Six et al. 2002). These non-ideal conditions for SOM decay are associated with physical and chemical protection mechanisms offered by the soil matrix that cither impede the access of enzymes to SOM (e.g. within microaggregates or by creating hydrophobicity) or increase the energy needed to degrade SOM through interactions with minerals (Eusterhues et al. 2003). In addition, enhanced SOM preservation may occur when environmental conditions are not adequate for microbial growth, e.g. the presence of free Al and Fe, low soil pH and/or low nutrient availability (Buurman and Roscoe 2011). Furthermore, intrinsic recalcitrance of specific SOM components may increase its longevity in soil. This is most likely the main process responsible for the long turnover time of highly condensed aromatic compounds present in BC (Harvey et al. 2012).

The nature of permanganate- and dichromate-oxidation resistant SOM is still poorly understood. Here we study the molecular properties of KMn[O.sub.4-] and [K.sub.2][Cr.sub.2][O.sub.7]-oxidationresistant SOM from a BC-rich colluvial soil by Py-GC/MS and solid-state [sup.13]C NMR, which may add to our knowledge on the stability of specific organic compounds against soil microbial oxidative enzymes.

Materials and methods

Study site and sample descriptions

Soil PRD-4 is a 2.4-m-thick Haplic Umbrisol (humic/alumic) according to 1USS Working Group WRB (2006) and Humic Pachic Dystrudept according to Soil Survey Staff (1998). This soil type is traditionally referred to as Atlantic Ranker (Carballas et al. 1967). Radiocarbon dating showed that the soil gradually accumulated through colluviation during the last ~13 000 years (Kaal et al. 2011). Soil PRD-4 has a deep black colour owing to a combination of high SOM content and abundance of BC (Table 1). For the present study, three samples were selected from this soil, corresponding to three periods with radically different ecosystems and, hypothetically, different SOM compositions. Sample SI (5-10cm depth) corresponds to recent material (<150 years BP), evidenced by [sup.14]C dating and the presence of pollen of exotic species Eucalyptus sp. (Lopez-Merino et al. 2012) (Table 1). This soil layer contains considerable amounts of 'fresh' (non- or slightly degraded) SOM and root fragments, as described by Kaal and van Mourik (2008). The vegetation corresponding to this sample is a mosaic of shrubland (dominated by Ericaceae), pasture and exotic tree species. Sample S2 (95-100 cm depth) contains large amounts of charcoal from palaeofires (Kaal et al. 2011) that occurred ~5000 years ago. Anthracological analysis showed that most charcoal originates from deciduous Quercus sp. This sample is thought to correspond to an oak-dominated woodland under substantial fire and grazing pressure. Sample S3 (190-195 cm depth) corresponds to an Early-Holocene phase (~9700 years BP) that preceded the colonisation of the area by deciduous forest, with 'steppe-like' vegetation dominated by Betula sp. (birch), shrubs of the Fabaceae family and herbaceous species.

Determination of organic C fractions

Potassium dichromate-oxidisable OC (O[C.sub.dichro]) was determined following the Waikley-Black oxidation method as modified by Wolbach and Anders (1989) and Knicker et al. (2007). Dry soil (0.5 g, <2 mm) was oxidised in triplicate with 20 mL of 0.2 M [K.sub.2][Cr.sub.2][O.sub.7] and 20 mL of concentrated [H.sub.2]S[O.sub.4] at 60[degrees]C in a water bath for 6 h. Control samples without soil were also analysed. After the reaction, excess dichromate was determined by titration against 0.033 M FeS[O.sub.4]. The amount of dichromate consumed by the soil was used to calculate the amount of dichromate-oxidisable OC (O[C.sub.dichro]) assuming that (i) the oxidation state of soil OC is zero ([C.sup.0]) and (if) complete oxidation to [C.sup.+4] occurs.

Potassium permanganate-oxidisable organic C (O[C.sub.per]) was determined, in triplicate, using 25 mL of 33 mM KMn[O.sub.4] solution added in 50-mL centrifuge tubes containing an amount of dry soil (<2 mm) equivalent to 15 mg organic C (Tirol-Padre and Ladha 2004). After 24 h shaking, the tubes were centrifuged for 5 min at 2600G and the supernatant diluted in distilled water (1:25v/v). Absorbance was read on a split-beam spectrophotometer at 565 nm. Blanks and a standard soil were analysed before each run. For calculation purposes, it was assumed that three moles of C (e.g. carbohydrates) are oxidised for every four moles of [Mn.sup.+7] reduced (Tirol-Padre and Ladha 2004).

Isolation of SOM fractions

The non-oxidised (NO) SOM extraction was considered the control treatment, consisting of SOM extraction by NaOH as described by Buurman et al. (2007). Briefly, 5 g soil (air- dried fine earth <2 mm) was extracted with 50 mL of 1 M NaOH and shaken for 24 h under [N.sub.2] to prevent oxidation/saponification. The suspension was centrifuged at 2600G for 1 h and the extract decanted, after which the extraction was repeated. The two extracts were combined and the residues discarded. The extracts were then acidified to pH 1 with concentrated HCl to protonate SOM. One mL of concentrated HF was added to dissolve silicates and increase C content of the extracted fraction. The acid mixture was shaken for 48 h, after which it was dialysed to neutral pH against distilled water to remove excess salt. Finally, the suspension was freeze-dried.

Dichromate-oxidation-resistant SOM(CR)

Soil (12 g) was oxidised in 500 mL of 0.2 M [K.sub.2][Cr.sub.2][O.sub.7] and 100 mL of concentrated [H.sub.2]S[O.sub.4] for 6h at 60[degrees]C using a water bath. Once cooled, the suspension was centrifuged at 2600G for 1 h and the supernatant decanted, after which the sediment was washed with distilled water until the solution was colourless. The suspension was discarded and the dichromate oxidation-resistant SOM extracted by 200 mL of 1 M NaOH for 24 h under [N.sub.2]. The resultant suspension was centrifuged at 2600G for 1 h and the supernatant decanted. This extraction was repeated twice. Thereafter, the three extracts were combined and acidified to pH 1 with concentrated HCl. One mL of concentrated HF was added to dissolve silicates and increase the content of organic C of the extracted fraction. This acid mixture was shaken for 48 h, dialysed against [H.sub.2]O to neutral pH and finally freeze-dried.

Permanganate-oxidation-resistant SOM (MN)

A volume of 1000mL of 33 mM KMn[O.sub.4] was added to 4.8, 8.2 and 7.0g dry soil (<2mm) for samples S1, S2 and S3, respectively, aiming to add 25 mL of KMn[O.sub.4] per 15 mg of organic C (calculated from O[C.sub.dichro] values). After 24 h shaking, the suspension was centrifuged at 2600(7 for 1.5 h and the extract decanted, after which the sediment was washed with distilled water until the supernatant was colourless. The MN in the residue was isolated and purified using 200 mL of 1 m NaOH for 24 h under [N.sub.2] atmosphere, analogous to the extraction of CR described above.

Py-GC/MS

Platinum filament Py-GC-MS was performed with a Pyroprobe 5000 (CDS Analytical Inc., Oxford, PA, USA) coupled to a 6890 GC and 5975 MS (Agilent Technologies, Palo Alto, CA, USA). The non-oxidised (NO) and oxidation-resistant SOM fractions (MN and CR) were pyrolysed at 750[degrees]C for 10 s (heating rate 10[degrees]C/ms). Analyses of sample S2 of the CR series was repeated, first at 400[degrees]C and then at 750[degrees]C, to distinguish between evaporation and pyrolysis products from volatile and macromolecular components, respectively. The pyrolysis interface was set at 300[degrees]C and the GC inlet at 325[degrees]C. The oven of the GC was heated from 50 to 325[degrees]C at 10[degrees]C/min and held isothermal for 5 min. The GC/MS transfer line was held at 325[degrees]C, the ion source (in electron impact mode, 70 eV) at 230[degrees]C and the quadrupole detector at 150[degrees]C, measuring fragments in the m/z 50-500 range. The GC was equipped with a (non-polar) HP-1 100% dimethylpolysiloxane column. Helium was used as the carrier gas (constant gas flow, 1 mL [min.sup.-1]). The major peaks in the total ion current of all samples were listed and, if possible, identified using the NIST 05 library and Py-GC/MS literature (Appendix A). Quantification of these pyrolysis products, 172 in total, was obtained by using the peak area of the major fragment ions (m/z). The sum of these peaks, i.e. total quantified peak area (TQPA), was set as 100%, and the relative proportions of the pyrolysis products were calculated as the percentage of TQPA. This semi-quantitative estimate allows for better comparison among samples than visual inspection of pyrolysis chromatograms (pyrograms) alone, and produces a dataset that can be treated statistically.

[sup.13]C NMR spectroscopy

Solid-state NMR spectroscopy experiments were performed with cross-polarisation magic angle spinning (CP MAS) at 298 K. in a 17.6 T Varian lnova-750 spectrometer (operating at 750 MHz proton frequency) equipped with a T3 Varian solid probe (Varian Inc., Palo Alto, CA, USA). Solid NMR samples were prepared in 3.2-mm rotors with an effective sample capacity of 22 [micro]L, which corresponds to ~30mg of the powdered sample. Spectra were processed and analysed with MestreC software (Mestrelab Research Inc., Santiago de Compostela, Spain). Carbon chemical shifts were referred to the carbon methylene signal of solid adamantane at 28.92 ppm. This sample was also used for the calibration of the 1D CP MAS experiments. The ID CP MAS spectra were acquired for the samples with the following conditions: the inter-scan delay was set to 0.5 s, the number of scans was 24000 and the MAS rate was 20 kHz. Heteronuclear decoupling during acquisition of the FID was performed with Spinal-64 with a proton field strength of 70 kHz. The cross-polarisation time was set to 1 ms. During cross-polarisation, the field strength of the proton pulse was set constant to 75 kHz and that of the [sup.13]C pulse was linearly ramped with a 20-kHz ramp near the matching sideband. Spectra were divided into different regions of chemical shift following Knicker et al. (2005). Relative abundances of the various C groups were determined by integration of the signal intensity in their respective chemical shift regions. The region 0-45 ppm is assigned to alkyl C corresponding to terminal methyl groups and methylene groups of aliphatic moieties. The O-alkyl C region, typically assigned to carbohydrate-derived structures, is 45-95 ppm. Here, between 45 and 60 ppm, N-alkyl C (i.e. in amino sugars and peptide structures) can contribute to the signal. Between 90 and 160 ppm, resonance lines of olefins and aromatic C are detected. The regions from 160 to 220 ppm and from 220 to 245 are assigned to carbonyl C separated into carboxyl/amide and aldehyde/ketone groups, respectively. Although it is often assumed that solid-state [sup.13]C NMR underestimate BC, recent studies demonstrated that most charcoals have an atomic H/C ratio >0.5 and thus provide sufficient protonation for efficient cross-polarisation and reliable NMR spectra (Knicker et al. 2005). Because of the limited sample availability, some samples required Al-oxide to fill the rotor, causing some signal quality deterioration. Samples MN-2, MN-3 and CR-3 were not analysed for that reason.

Factor analysis

The relative proportions of pyrolysis products were subjected to factor analysis using Statistica Version 8 (StatSoft, Tulsa, OK, USA). Factor analysis proved useful in the interpretation of Py-GC/MS datasets, especially with respect to the sources and degradation states to which the pyrolysis products correspond.

Results and discussion

Py-GC/MS: source allocation

The pyrolysis products were grouped according to their chemical structure into the following classes: (i) aliphatic compounds (homologous scries of n-alkanes and n-alkenes, and branched alkencs), (ii) lignin-derived methoxyphenols, (iii) phenols, (iv) monocyclic aromatic compounds (MAHs), (v) polycyclic aromatic hydrocarbons (PAHs), (vi) N-containing compounds, (vii) carbohydrate-derived pyrolysis products, and (viii) unidentified compounds. Appendix A is a list of the pyrolysis products identified.

Aliphatic compounds

The n-alkane/n-alkene pairs, ranging from [C.sub.10] to [C.sub.28], originate largely from aliphatic biopolymers (Eglinton and Hamilton 1967). The other short- and mid-chain (~[C.sub.10]-[C.sub.20]) alkenes, not from the homologous scries and most of which are probably branched, are considered significant products of charred aliphatic matter according to recent studies, even though they are not produced exclusively from pyrolysis of BC (Eckmeier and Wiesenberg 2009; Kaal et al. 2012a). Several n-fatty acids (mainly [C.sub.16] and C,8) seemed to increase disproportionally upon chemical oxidation, especially in CR. This might be explained by the enrichment of aliphatic structures upon dichromatc oxidation due to their hydrophobicity (Knicker et al. 2007). However, the use of a HP-1 column, which has a larger internal diameter than is frequently used for non-polar columns for Py-GC/MS, may have affected the 'chromatographic mobility' and the relative proportions of these compounds, thus making its interpretation difficult. Therefore, these compounds were not included in the statistical analyses.

Lignin-derived methoxyphenols

Methoxyphenols (guaiacyl- and syringyl-based) are typical products of coniferyl and sinapyl lignin, respectively (Boeijan et al. 2003); 4-vinylphenol was also added to this group because it has frequently been shown to be marker of coumaryl lignin and the non-lignin coumaric acid in grasses (Saiz-Jimenez and de Leeuw 1986). An unknown proportion of 4-vinylphenol and 4-vinylguaiacol may originate from non-lignin phenolic acids as well (Schellekens et al. 2012), but that does not influence the interpretation of results here as statistically they behave as the lignin markers (see below).

Phenols

The other phenols have multiple origins. Phenol and [C.sub.1]-[C.sub.2]-alkylphenols may originate from any phenolic precursor including lignin, tannin, proteinaceous biomass, weakly charred BC and carbohydrates (Tegelaar et al. 1995; Stuczynski et al. 1997), whereas lignin, tannin or thermally demethylated lignin (Kaal et al. 20126) are the most likely precursors of 1,2-bcnzenediol (catechol).

Monocyclic aromatic hydrocarbons

The MAHs include benzene, toluene, styrene, dimethylbenzenes, linear [C.sub.2]-[C.sub.4]-alkylbcnzenes, a dimethylstyrene and a dimethyl-methylethylbenzenc compound. They are formed from many aromatic and some non-aromatic precursors (Schulten et al. 1991), but BC is known to produce an exceptionally high proportion of benzene (Kaal et al. 2012a). Indeed, the analysis of the products of incomplete combustion by Py-GC/MS showed that MAHs and PAHs are major pyrolysis products of BC (Pastorova et al. 1994; Almcndros et al. 2003). Renewed interest in the detection of burning residues in SOM has established that BC is a major source of SOM and that its oxidation products could be a potential source of highly aromatic humic acids (Hatcher et al. 1989; Skjemstad et al. 1996; Shindo et al. 2004). On the other hand, the alkylstyrenes most likely originate from the monoterpenes present in Eucalyptus globulus litter (see below).

Polycyclic aromatic hydrocarbon compounds

The origin of PAHs in SOM pyrolyzates have been the subject of considerable debate. They were sometimes considered actual building blocks of humic substances formed upon condensation reactions during humification (Schulten et al. 1991), or more frequently interpreted as analytical artefacts because of evidence of their formation during pyrolysis of aliphatic compounds through cyclisation and aromatisation (Saiz-Jimenez 1994/?). More recently, PAHs and particularly the non-alkyl-substituted and >2 ring PAHs (Rumpel et al. 2007) are considered indicative of (but not markers of) BC in SOM (Kaal and Rumpel 2009; Song and Peng 2010). In the present study, unsubstituted PAHs (indene, naphthalene, fluorene, biphenyl, phenanthrene and anthracene) and [C.sub.1]-[C.sub.2] alkyl analogues of these PAHs were abundant. In addition, a series of [C.sub.3]-[C.sub.4] alkylnaphthalenes and [C.sub.5:0] and [C.sub.5:1] alkylnaphthalenes probably originate from evaporation and pyrolysis of monoterpenes (e.g. pinene, phellandrene, eucalyptol) and sesquiterpenoids (aromadendrene, globulol), respectively, present in Eucalyptus sp. oil. Indeed, these compounds were identified in Eucalyptus globulus litter (a mixture of leaves, cortex and branches) pyrolyzates (data not shown). Some of the polysubstituted PAHs ([C.sub.3]-indene, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and [C.sub.5:1]-naphthalene) provided the largest contributions to the pyrograms of sample SI (contrary to many unsubstituted or [C.sub.1] -alkyl-substituted PAHs, largely from BC); this suggests that a significant portion of these compounds originate from fresh Eucalyptus sp. litter. Overall, it should be noted that, in this study, where distinguishing between BC and non-pyrogenic SOM components will appear to be important, the interpretation of MAHs and PAHs relies more strongly on the growing body of knowledge on BC's pyrolysis fingerprints (Fabbri et al. 2012; Kaal et al. 2012a; Song and Peng 2010) than on previous studies on the structural characteristics of humic acids (Schulten et al. 1991; Saiz-Jimenez 1994a).

Products containing N

Of the 24 N-containing pyrolysis products identified, benzonitrile and Ci-benzonitriles were recently proposed as the main products of N-containing groups in BC (Schnitzer et al. 2007; Song and Peng 2010). In addition, isoquinoline, phenylpyridine, benzenedicarbonitriles and pyridinecarbonitriles can be considered as markers of 'black N' (BN) (Knicker 2007; Kaal et al. 2009). Note that absence of these products does not imply absence of BN; these compounds can probably only be detected in high-quality pyrograms of exceptionally BN-rich samples. Pyrroles, pyridines and indoles are potential products of BN as well, but these compounds are common in the pyrolyzates of non-pyrogenic N-moieties. Several markers of chitin (acetamide and a compound tentatively identified as trianhydro-2-acetamido-2deoxyglucose; Van der Kaaden et al. 1984; Stankiewicz et al. 1996) and chitin-entangled protein (diketopiperazine) probably originate from fungal cell walls and/or arthropod exoskeleta, cither way serving as an indication of biologically re-assimilated ('secondary') remains in SOM (Gutierrez et al. 1995). Finally, for picolinamide, cyanobenzoic acid and phthalimide-based compounds, no specific origin has been identified yet.

Carbohydrate compounds

Of the carbohydrate products identified, levoglucosan, dianhydro-[alpha]-glucopyranose, pyranones and dianhydrorhamnose largely originate from 'fresh' or well-preserved polysaccharides (Stuczynski et al. 1997; Poirier et al. 2005; Nierop et al. 2005). On the other hand, cyclopentenediones, furans, furfurals, levoglucosenone and dibenzofuran originate from fresh and/or degraded carbohydrates (Buurman and Roscoe 2011). This degradation may be either biological or thermal in nature, the latter especially for the furans, furaldehydes and dibenzofuran (Pastorova et al. 1994; Boon et al. 1994).

Unidentified compounds

An unsaturated non-aromatic cyclic compound (Ul) was identified only in the pyrolyzates from SI (NO-1 and MN-1), which also contained the pollen of Eucalyptus sp. It probably corresponds to a-phellandrene, which is abundant in eucalyptus oils (Samate et al. 1998). Furthermore, several polymethyl-substituted polycyclic compounds (U3-U6), also detected in the aforementioned fresh eucalyptus-litter pyrolyzatc, probably derived from Eucalyptus sp. Finally, a methylated cyclohexane (U2) of unknown origin was tentatively identified.

Py-GC/MS: quantification and interpretation

Pyrograms of chemical oxidation resistant SOM fractions and non-oxidised samples are represented in Fig. 1. The relative contributions to TQPA for identified groups in the different soil horizons studied are presented in Table 2. In NO-1, carbohydrate-derived pyrolysis products accounted for 30% of TQPA, with levoglucosan (Ps13) from intact polysaccharide (Stuczynski et al. 1997; Poirier et al. 2005) being dominant. The presence of 4-hydroxy-5,6-dihydro(2H)-pyranone (Ps6) and dianhydrorhamnose (Ps7) confirms the existence of fresh (or well preserved) polysaccharides in NO-1 (Nierop et al. 2005). Of the samples studied, these compounds showed the largest contribution to sample NO-1. The same pattern was observed for many other indicators of fresh plant material, including the aliphatic compound producing m/z 83+280, diketodipyrrole, and the lignin-derived products (Suarez-Abelenda et al. 2011). The large proportion of phenols in the pyrolyzate of this sample may be explained by the abundance of lignin. Samples NO-1 and MN-1 had the highest contributions of probably eucalyptus-derived moieties ([C.sub.5:0]-, [C.sub.5:1]-alkylnaphthalenes and a phellandrene and [C.sub.3]-naphthalenes). It is concluded that the SOM of NO-1 is characterised by a large fraction of well-preserved polysaccharides and lignin, with an additional contribution of specific eucalyptus-derived substances, and relatively small proportions of microbial and pyrogenic SOM. The latter is supported by the low benzcne/alkylbenzenes and PAH/alkyl-PAHs ratios (Table 2), which are indicative of a low contribution of strongly charred BC to the MAHs and PAHs of these samples (Kaal and Rumpel 2009; Kaal et al. 2012a).

Unsurprisingly, sample NO-2 (~5000 years old), produced fewer pyrolysis products from fresh SOM than NO-1. More specifically, in comparison with NO-1, among the carbohydrate markers there was a strong increase of furans, furaldehydes, levoglucosenone and acetic acid, while levoglucosan, pyranones and dianhydrorhamnose diminished, which is a clear indication of a shift of fresh polysaccharide to degraded/microbial carbohydrates (Saiz-Jimenez and de Leeuw 1986; Buurman and Roscoe 2011). Lignin markers were virtually absent. The NO-2 sample produced many N-compounds, including those from chitin (N3 and N22), pyridine (N1, often associated with microbial SOM; Buurman et al. 2007) and BN (e.g. aromatic carbonitriles and phcnylpyridine). It also gave higher proportions of MAHs and BC-derived PAHs than the NO-1 sample. It is concluded that the SOM of sample NO-2 was predominantly composed of degraded/microbial and pyrogenic material.

The pyrolyzate of sample NO-3 was dominated by carbohydrate markers, with acetic acid, 3/2-furaldehyde, 5methyl-2-furaldehyde, dianhydro-a-glucopyranose, a furanone and 4-acetylfuran accounting for 62% of TQPA (Table 2). These pyrolysis products are frequently ascribed to SOM with large proportions of microbial biomass (Saiz-Jimenez and de Leeuw 1986; Buurman and Roscoe 2011). The small relative proportions of MAHs and PAHs suggest that BC accounts for only a minor portion of the SOM in NO-3. This is supported by the low ratios of benzene/alkyl-benzenes and PAH/alkyl-PAH (Table 2).

In general, the differences in pyrolyzate compositions between NO-samples and MN-samples were small, yet some are worth mentioning. For sample S1, oxidation with KMn[O.sub.4] (which promoted a decrease of 2.3 mg [g.sup.-1] of OC; Table 1) caused an increase in MAHs (from 16.4% in NO-1 to 30.0% in MN-1) and decrease in carbohydrates (from 30.4% to 18.4%) and lignin (from 7.4% to 3.0%) (Table 2). These results can be explained by the partial oxidation of fresh SOM (van Soest and Wine 1986; Tirol-Padre and Ladha 2004) and the relative enrichment of pyrogenic (Almendros et al. 1990) and aliphatic SOM (Gonzalez-Vila and Martin 1985; Almendros et al. 1989). Permanganate oxidation concentrates pyrolysis products from aromatic structures present in humic acids in general (Polvillo et al. 2009), even though cyclisation of aliphatic precursors may also play a role (Gonzalez-Vila and Martin 1985). In sample S2, KMn[O.sub.4] oxidation (MN-2) (with a smaller decrease of OC, 1.3 mg [g.sup.-1]) caused a strong decline in carbohydrate products (from 31.5% of TQPA in NO-2 to 17.4% in MN-2) and an increase in aliphatic pyrolysis products (sum of n-alkanes, n-alkenes and other aliphatic compounds from 5.3% in NO-2 to 23.1% in MN-2). These changes are indicative of selective oxidation of (an unknown proportion of) the degraded/ microbial SOM and the relative enrichment of aliphatic precursors probably from degraded root components (Kaal and van Mourik 2008). In addition, MN-2 produced higher amounts of N-containing BC markers, as BC and BN are relatively resistant against oxidation with this reagent. Skjemstad et al. (2006) found that KMn[O.sub.4] may react significantly with BC, but no evidence of this was found here. Finally, sample MN-3 (with a decrease of 1.7 mg [g.sup.-1] of its OC content) contains a smaller proportion of carbohydrates than NO-3 (17.7% v. 62.2% of TQPA), confirming that the degraded/microbial carbohydrate fraction is relatively susceptible to this oxidation agent.

The pyrolyzates obtained from the residues after dichromatc oxidation were very different from those of NO-and MN-samples. The CR-1 sample (27.5 mg [g.sup.-1] of its OC was oxidised by dichromate; Table 1) was strongly enriched in aliphatic pyrolysis products (34% of TQPA), particularly of short-chain (<[C.sub.18]) n-alkanes/n-alkenes (located on the right side of the broken line in the aliphatic cluster; SE quadrant, Fig. 2) and branched alkenes, and depleted in lignin-, carbohydrateand eucalyptus-derived pyrolysis products in comparison with NO-1 and MN-1. The CR-I sample also produced the highest proportions of 3-ring PAHs and higher ratios of benzene/alkylbenzenes and PAH/alkyl-PAHs ratios (Table 2), suggesting that large proportions of the MAHs and PAHs from the CR-1 are pyrogenic. These results are indicative of the enrichment of pyrogenic SOM in the fraction resistant to [K.sub.2][Cr.sub.2][O.sub.7]-oxidation. Indeed, the partial resistance of BC to cc oxidation is well documented (Knicker et al. 2007, 2008). The same studies showed the existence of a [K.sub.2][Cr.sub.2][O.sub.7]-resistant alkyl fraction, which was also supported by the pyrolyzate composition of CR-1. The increase was also observed for the n-fatty acids (data not shown), which are not considered part of structural aliphatic plant material. These results support the hypothesis that the enrichment of aliphatic material in the residual fraction of the [K.sub.2][Cr.sub.2][O.sub.7] oxidation residues is produced by the hydrophobic nature of these constituents (Knicker et al. 2007), possibly in combination with chemical recalcitrance of C-C bonds in methylene chains. Finally, a decrease was observed for the intact terpene-like plant-derived PAHs, clearly showing different origin for the unsubstituted and methyl-substituted PAHs (mainly from BC) and the polyalkyl-substituted PAHs from eucalyptus litter. For sample CR-2, dichromatc oxidation oxidised less OC (12.3 mg g 1 for CR-2) than for CR-1. It produced a further decrease in the proportion of lignin markers in comparison with MN-2, and of microbial products such as acetamide and fiirans, while the BC and BN fingerprints were relatively intense (e.g. benzene, unsubstituted PAHs, benzene carbonitriles, isoquinoline and dibenzofuran). The largest proportion of these BC-derivcd pyrolysis products coincides with the highest macroscopic charcoal content of sample S2 (Table 1). These results confirm the accumulation of BC and BN in the residues after [K.sub.2][Cr.sub.2][O.sub.7]v oxidation. Similar to CR-1, sample CR-2 was enriched in aliphatic pyrolysis products. Unexpectedly, significant amounts of the markers of well-preserved polysaccharides such as levoglucosan were detected in the pyrolyzates of CR-2 and CR-3. With the information available at this moment, our best explanation to this observation is the presence of an uncharred cellulose-containing core (Knicker et al. 2005) in incompletely charred particles and thereby protected against [K.sub.2][Cr.sub.2][O.sub.7] oxidation. Sample CR-3 (in which dichromate caused a loss of 14.3 mg [g.sup.-1] of OC) was also enriched in pyrogenic SOM with a high contribution of BN markers, and in an aliphatic component with particularly high contributions of branched alkenes from an aliphatic SOM fraction, possibly in part pyrogenic.

Py-GC/MS: factor analysis

The first four factors (F1-F4) explained 81% of the variation in the Py-GC/MS dataset, with FI and F2 combined accounting for 61 %. The loadings of the pyrolysis products, and the scores of the samples analysed, are shown in F1-F2 factor space (Fig. 2).

The n-alkanes/n-alkenes are predominantly represented in the SE quadrant, with high positive loadings on F1. Lower loadings on FI were observed for the < -alkenes > [C.sub.20] than for [C.sub.10]-[C.sub.20] n-alkenes. Branched alkenes plot between their straight-chain analogues and the pyrogenic SOM markers, supporting the hypothesis that these branched alkenes are associated with charred aliphatic precursors (Eckmeier and Wiesenberg 2009; Kaal and Rumpel 2009).

The N-containing compounds are spread throughout the F1-F2 factor space, which is a result of the diverse origin of the members of this group. One cluster of N-containing compounds in the NE quadrant is composed of benzonitrile, [C.sub.1]-ben/onitriies, benzene dicarbonitriles, cyanobenzoic acid, pyridine, [C.sub.1]-pyridine and pyridinecarbonitrile, clearly reflecting a pyrogenic origin. Indeed, many non-alkylsubstituted PAHs and dibenzofuran, also associated with BC (Pastorova et al. 1994), plot in the same region. Chitin-derived N compounds (chitin markers such as acetamide) and 4-acetylfuran are spread out in the NW quadrant together with microbial polysaccharides, whilst diketodipyrrole denotes the presence of fresh SOM in the SW quadrant (see below). Indoles are spread throughout the SW and SE quadrants, which may be indicative of a mixed origin.

The lignin-derived products (including free phenolic acids), i. e. 4-vinylphenol, guaiacols and syringols), and catechol occur in the SW quadrant together with an aliphatic marker of fresh plant material (Al13). Most of the remaining pyrolysis products in this region are associated with fresh or well-preserved SOM components as well: phenol and alkylphenols (in this case from lignin), [C.sub.5:0] and [C.sub.5:1] alkylnaphthalenes (from eucalyptus litter), dianhydrorhamnose (from polysaccharides), and diketodipyrrole and indoles (typical N-containing pyrolysis products of fresh proteinaceous biomass; Buurman and Roscoe 2011). Levoglucosan plots between fresh OM and charred material; this may be attributed to the aforementioned protection of cellulose in interior parts of charcoal particles in dichromate oxidation residues or an unknown alternative levoglucosan source. The other carbohydrate products are spread along F2 because they have multiple sources, most of which corresponding to degraded/microbial SOM. The

carbohydrate products of dcgraded/microbial SOM are probably those that plot in the NW quadrant: furans (4-acetylfuran, 3/2furaldehyde and 5-methyl-2-furaldehyde), glucopyranose, which is a microbial marker (Nierop et al. 2005), and acetic acid. This interpretation is consistent with the presence of the markers of chitin in this region of factor space.

In summary, F1-F2 separates the pyrolysis products according to their principal origin. Factor 1 separates the pyrogenic and aliphatic oxidation-resistant SOM fractions (chemically stable) from the fresh and degraded SOM fractions (chemically labile), while decomposed and pyrogenic SOM (strongly altered) are separated from fresh and oxidation-resistant aliphatics (resembling plant material) according to their loadings on F2.

The factor scores of the samples can be used to identify the main differences between the samples analysed (Fig. 2). As such, the samples with a large fraction of fresh biomass (NO-1 and MN-1) plot in the SW quadrant. Sample CR-1 plots in the SE quadrant, as dichromate oxidation eliminated most of the fresh SOM, causing the relative accumulation of aliphatic SOM and weakly charred material. Sample S2 is a mixture of mainly degraded SOM and BC (with small contributions of fresh and aliphatic material), which is why NO-2 plots in the NW region, dominated by degraded/microbial markers, whereas MN-2 and CR-2 plot in the NE quadrant because of relative enrichment of BC after chemical treatment. Sample S3 was also rich in microbial SOM but has a lower content of chemically recalcitrant/hydrophobic aliphatic SOM and BC, and higher proportion of degraded/microbial SOM. This explains why NO-3 has a high F2 score while MN-3 and CR-3 plot in the NE quadrant, reflecting BC enrichment after the selective depletion of degraded/microbial SOM. The short distance in factorial space between MN-3 and CR-3, and the large distance between these samples and NO-3, suggests a that the abundant degraded/microbial biomass (carbohydrates, chitin) in this sample is highly susceptible to permanganate and dichromate treatment.

From these results, some inferences on the effects of the oxidation agents on SOM composition can be made. First, the minor differences between NO-1 and MN-1 can be explained by the relatively small microbial contribution to sample SI. By contrast, [K.sub.2][Cr.sub.2][O.sub.7] thoroughly modified the pyrolysis fingerprint obtained from the residues of SI by eliminating lignin, polysaccharides and terpenes, and relative enrichment of aliphatic and pyrogenic structures. In the older samples, where the aliphatic fraction is less dominant while that of degraded/microbial and pyrogenic SOM prevail, chemically oxidised samples (MN-2, MN-3, CR-2 and CR-3) had positive scores on F1 and F2 mainly because both oxidants concentrate BC, with [K.sub.2][Cr.sub.2][O.sub.7] being the stronger oxidant.

Solid-state [sup.13]C NMR spectroscopy: results and comparison with Py-GC/MS

Samples are compared by relative intensity of the chemical shift regions. The spectrum of NO-1 (Fig. 3) was characterised by a dominant signal at 21 ppm and a shoulder at 29 ppm (combined 29%, Table 3) from alkyl C, which can be ascribed to aliphatic structures in fatty acids, lipids, waxes, cutan, suberan, cutin and suberin (Tegclaar et al. 1989) but also peptide structures and short alkyl side-chains. In the O-alkyl C region (45-110 ppm) a broad peak at 75 ppm (with a contribution of 29%) was detected, which is generally attributed to cellulose, hemicelluloses and pectins (Gramble et al. 1994; Kogel-Knabner 1997). The peak at 55 ppm probably corresponds to methoxyl groups in lignin structures (Kogel-Knabner 1997) but can also have contributions of (V-alkyl from amino sugars and peptides. The O-substituted aromatic C between 140 and 160 ppm may derive from lignin and oxidised BC (Knickcr et al. 2005). Resonance lines of aromatic C-H groups are detected in the chemical shift region between 110 and 140 ppm (14%) (Knicker and Ludemann 1995). The chemical shifts of carbon in carboxylic acids, esters and amides fall within the range 160-220 ppm, and represents 15% of the total [sup.13]C intensity. There are minor contributions of carbonyl or aldehydes, giving signals between 220 and 245 ppm.

The NMR spectra of NO-2 and NO-3 differ considerably from that of NO-1. In NO-2, the O-alkyl C fraction has the highest values (26%), followed by alkyl C, carboxyFamidc C and aromatic C. Considering that there is no clear signal in the O-substituted C region (140-160 ppm), this spectrum can be best explained with a considerable contribution of oxidised charcoal. Sample NO-3 presented the highest contribution of non-lignin aromatic fraction, probably BC-derived (31%) and a large signal of carboxyl/amide C (26%), which likely originates from microbial compounds.

The chemical oxidants had several effects on the NMR signal obtained from sample SI. After the KMn[O.sub.4] treatment (MN-1), and comparable to results from Py-GC-MS, a spectrum similar to that of N O-l but with slightly higher relative intensities in the alkyl C (34%) region was acquired, confirming other [sup.13]C NMR studies (Tirol-Padre and Ladha 2004) in that cellulose is largely resistant to permanganate oxidation. Dichromate oxidation of sample SI caused an increase of the relative contribution of aromatic C (sum of aromatic C-H and aromatic C-O-R) (from 17% in NO-1 to 25% in CR-1) with a concomitant depletion of methoxyl C/Al-alkyl C and O-alkyl C (see Table 3). Note that differences between NO-1 and CR-1 by NMR are smaller than observed by Py-GC/MS. This indicates that, as Py-GC/MS data seem to be best supported, NMR results must be regarded with caution.

The spectrum of CR-2 showed the highest intensity in the chemical shift region of aromatic C (45%). Considering the absence of a methoxyl C signal, the width of the signal band (90-140 ppm) and the composition of the pyrolysis fingerprint, this aromatic signal originates from BC (see also Skjemstad et al. 1996; Knicker et al. 2005). Moreover, the contribution of alkyl C was strongly reduced upon K2Cr207 oxidation (10% in CR-2).

In summary, in NO samples, [sup.13]C NMR spectroscopy shows a relative decrease with depth of aliphatic C, carbohydrates and lignin moieties, and a relative increase with depth of a non-lignin aromatic fraction (probably BC) and carboxyl/amide C (possibly oxidised BC in combination with N-rich microbial SOM), which is in agreement with Py-GC/MS data. With regard to the effects of chemical oxidation, both Py-GC/MS and [sup.13]C NMR spectroscopy showed a decrease of easily degradable SOM (mainly composed of fresh lignin and polysaccharides) and an increase of the aromatic fraction. Furthermore, lignin was slightly oxidised by KMn[O.sub.4], contrary to that observed in previous studies (van Soest and Wine 1986; Tirol-Padre and Ladha 2004; Skjemstad et al. 2006), where lignin was strongly degraded. The increase of aliphatic moieties upon chemical oxidation, as suggested by Py-GC/MS, was also observed by NMR spectroscopy in the superficial sample SI, although it strongly decreased in S2. However, here one must bear in mind that the alkyl C region not only contains an intensity of lipids but also has considerable contributions of peptide structures or short alkyl side-chains (such as C3-side chains in lignin). Those moieties may be expected to be relatively susceptible to oxidation resulting in a relative depletion of the signal intensity in the alkyl C region even though longer chain aliphatic components may have experienced a relative enrichment.

Conclusions

The molecular study of SOM fractions of three horizons of a colluvial soil representing ages of 100, 5000 and 9700 years, before and after treatment with KMn[O.sub.4] and [K.sub.2][Cr.sub.2][O.sub.7], provided detailed information on SOM composition (with regard to source and degradation/preservation state) and the behaviour of different SOM fractions towards these oxidation agents. Eucalyptus-derived terpenes and sesquiterpenes were only present in the youngest sample and resisted KMn[O.sub.4] but not [K.sub.2][Cr.sub.2][O.sub.7] oxidation. Microbial/degraded SOM, mostly composed of carbohydrates and chitin, was especially abundant in the deeper layers of the soil and appeared highly susceptible to both KMn[O.sub.4] and [K.sub.2][Cr.sub.2][O.sub.7] oxidation. As such, KMn[O.sub.4] could be used as an indication of the abundance of microbial biomass. Both oxidants, [K.sub.2][Cr.sub.2][O.sub.7] in particular, concentrated two other SOM fractions abundant in this soil: aliphatic and pyrogenic material (BC), the latter having a significant amount of N-containing functional groups (BN). These fractions probably survived [K.sub.2][Cr.sub.2][O.sub.7] oxidation because of the chemical stability of polyaromatic moieties (BC) and resistant C-C bonds in methylene chains and/or hydrophobicity of the aliphatic fraction (which is probably root-derived). It appeared that especially [K.sub.2][Cr.sub.2][O.sub.7] oxidation efficiently concentrates BC and oxidation-resistant aliphatic structures from other SOM sources, and that in combination with PyGC/MS it is possible to distinguish between these sources (yet not quantitatively) while [sup.13]C NMR may assist in obtaining estimations of their relative proportions. Finally, BC isolation by dichromate oxidation and posterior quantification through total digestion (Knicker et al. 2007) is discouraged as a significant aliphatic fraction resists dichromate producing an overestimation of its contents.

http://dx.doi.org/10.1071/SR13195

Appendix A. Pyrolysis product list, molecular mass
(M+), fragment ions used for quantification and
retention times relative to guaiacol (RT)

code        Name                        [M.sup.+]      mass

10:1-28:1   C10-28 alkene                140-392       55+69
10:0-28:0   C10-28 alkane                142-394       57+71
Al1         aliphatic compound            n.d.         55+70
Al2         alkane/anal or                n.d.       57+69+70
              methylated
              alkanol
Al3         branched alkene               n.d.         55+69
Al4         branched alkene               n.d.         55+69
Al5         branched alkene               n.d.         55+69
Al6         branched alkene               n.d.         55+69
Al7         alkene                        n.d.         55+69
Al8         alkene                        n.d.         55+69
Al9         branched alkene               n.d.         55+69
Al10        branched alkene               n.d.         55+69
Al11        alkene                        n.d.         55+69
Al12        branched alkene               n.d.         55+69
Al13        aliphatic compound           83+280       83+280
Al14        branched alkene               n.d.         55+69
Al15        branched alkene               n.d.         55+69
Ph1         phenol                         94          66+94
Ph2         acetophenone                   120        77+105
Ph3         C1-phenol                      108        107+108
Ph4         C1-phenol                      108        107+108
Ph5         C2-phenol                      122        107+122
Ph6         C2-phenol                      122        107+122
Ph7         catechol                       110        110+64
Lg1         guaiacol                       124        109+124
Lg2         4-methylguaiacol               138        123+138
Lg3         4-vinylphenol                  120        120+91
Lg4         4-vinylguaiacol                150        135+150
Lg5         syringol                       154        154+139
Lg6         4-methylsyringol               168        153+168
Lg7         C3-guaiacol                    196        149+164
Lg8         Propenoic acid,                178        161+178
              3-(4-methoxyphenol)
Ar1         benzene                        78           78
Ar2         toluene                        92          91+92
Ar3         C2-benzene ethyl benzene       106        91+106
Ar4         C2-benzene                     106        91+106
              dimethyl benzene
Ar5         styrene                        104        78+104
Ar6         C2-benzene                     106        91+106
              dimethyl benzene
Ar7         C3-benzene                     120        105+120
Ar8         C4-benzene                     134        91+119
Ar9         C5-benzene                     148          133
              (dimethyl-methylethyl)
Ar10        C4:1 -benzene                  132        132+117
              (oc-dimethylstyrene)
Ar11        C7-benzene                     176         91+92
Ar12        branched alkyl-benzene         148        91+119
Pa1         C1-indene                      130        130+115
Pa2         naphthalene                    128          128
Pa3         C1-naphathalene                142        141+142
Pa4         C1-naphathalene                142        141+142
Pa5         biphenyl                       154          154
Pa6         C3-indene                      158        143+158
Pa7         C2-naphthalene                 156        141+156
Pa8         C3-naphthalene                 170        155+170
Pa9         C3-naphthalene                 170        155+170
Pa10        Fluorene                       166        165+166
Pa11        C3-naphthalene                 170        155+170
Pa12        C1 -Fluorene                   180        165+180
Pa13        9H-Fluoren-9-one               180        152+180
Pa14        C4-naphthalene                 184        169+184
Pa15        phenanthrene                   178          178
Pa16        anthracene                     178          178
Pa17        C5-naphthalene                 198        183+198
Pa18        C5-naphthalene                 198        183+198
              (or C2-azulene)
Pa19        C5:1-naphtalene                202        159+145
Pa20        C5-naphtalene                  204        147+162
Pa21        C5:1-naphtalene                202        159+202
Pa22        C5-naphtalene                  204        91+105
Pa23        C5-naphtalene                  204        105+133
Pa24        C5-naphtalene                  204        91+105
Pa25        C5-naphtalene                  204        91+105
Pa26        C5-naphtalene                  204        91+105
Pa27        C5-naphtalene                  204        173+189
Pa28        C4-naphtalene                  186        143+171
Pa29        C5:1-naphtalene                202        159+80
Pa30        C5:1-naphtalene                202        146+133
Pa31        C5:1-naphtalene                202        159+202
N1          pyridine                       79          52+79
N2          pyrrole                        67           67
N3          acetamide                      n.d          59
N4          C1-pyrrole                     81          80+81
N5          C1-pyrrole                     81          80+81
N6          C1-pyridine                    93          66+93
N7          benzonitrile                   103        76+103
N8          pyridinecarbonitrile           104        104+77
N9          C1-benzonitrile                117        90+117
N10         C1-benzonitrile                117        90+117
N11         1,3-benzenedicarbonitrile      128        101+128
N12         picolinamide                   122        79+122
N13         1,3-bcnzenedicarbonitrile      128        101+128
NI4         isoquinoline                   129          129
N15         indole                         117        90+117
N16         phthalamic acid                104        104+76
N17         1,3-benzenedicarbonitrile      128        101+128
N18         C1-indole                      131        130+131
N19         C1-pthalimide                  161        161+76
N20         cyanobenzoic acid              147        76+147
N21         phenylpyridine                 155        154+155
N22         chitin-derived compound        167        125+167
N23         diketodipyrrole                186        93+186
N24         diketopiperazine compound      194        70+194
Ps1         acetic acid                    60           60
Ps2         furanone compound              84          54+84
Ps3         3/2-furaldehyde                96          95196
Ps4         acetylfuran                    110        95+110
Ps5         5-methyl-2-furaldehyde         110        109+110
Ps6         4-hydroxy-5,6-                 114        58+114
              dihydro-(2H)-pyranone
Ps7         dianhydrorhamnose              128        113+128
Ps8         cyclopentenedione              112        69+112
              compound
Ps9         levoglucosenone                126         68+98
Ps10        3-hydroxy-2-methyl-            126        71+126
              4H-pyran-4-one
Ps11        dianhydro-[alpha],             144         57+69
              D-glucopyranose
Ps12        dibenzofuran                   168        168+139
Ps13        levoglucosan                   162         60+73
U1          alpha phellandrene             136       91+93+136
U2          U2 (possibly                   96          67+96
              methylcyclohexane)
U3          U3                             157        117+157
U4          U4                             200        185+200
U5          U5                             212        197+202
U6          U6                             203          203

                                           RT/
code        Name                        guaiacol

10:1-28:1   C10-28 alkene              0.832-3.965
10:0-28:0   C10-28 alkane              0.857-3.969
Al1         aliphatic compound            0.549
Al2         alkane/anal or                1.157
              methylated
              alkanol
Al3         branched alkene               1.160
Al4         branched alkene               1.573
Al5         branched alkene               1.590
Al6         branched alkene               1.610
Al7         alkene                        1.901
Al8         alkene                        2.301
Al9         branched alkene               2.428
Al10        branched alkene               2.941
Al11        alkene                        3.069
Al12        branched alkene               3.272
Al13        aliphatic compound            3.342
Al14        branched alkene               3.565
Al15        branched alkene               3.799
Ph1         phenol                        0.782
Ph2         acetophenone                  0.937
Ph3         C1-phenol                     0.941
Ph4         C1-phenol                     0.991
Ph5         C2-phenol                     1.158
Ph6         C2-phenol                     1.202
Ph7         catechol                      1.348
Lg1         guaiacol                      1.000
Lg2         4-methylguaiacol              1.245
Lg3         4-vinylphenol                 1.326
Lg4         4-vinylguaiacol               1.519
Lg5         syringol                      1.585
Lg6         4-methylsyringol              1.798
Lg7         C3-guaiacol                   1.815
Lg8         Propenoic acid,               3.296
              3-(4-methoxyphenol)
Ar1         benzene                       0.347
Ar2         toluene                       0.431
Ar3         C2-benzene ethyl benzene      0.559
Ar4         C2-benzene                    0.574
              dimethyl benzene
Ar5         styrene                       0.605
Ar6         C2-benzene                    0.612
              dimethyl benzene
Ar7         C3-benzene                    0.815
Ar8         C4-benzene                    0.887
Ar9         C5-benzene                    1.160
              (dimethyl-methylethyl)
Ar10        C4:1 -benzene                 1.026
              (oc-dimethylstyrene)
Ar11        C7-benzene                    1.662
Ar12        branched alkyl-benzene        1.194
Pa1         C1 -indene                    1.146
Pa2         naphthalene                   1.231
Pa3         C1-naphathalene               1.491
Pa4         Cl-naphathalene               1.523
Pa5         biphenyl                      1.668
Pa6         C3-indene                     1.723
Pa7         C2-naphthalene                1.772
Pa8         C3-naphthalene                2.052
Pa9         C3-naphthalene                2.056
Pa10        Fluorene                      2.090
Pa11        C3-naphthalene                2.103
Pa12        C1 -Fluorene                  2.314
Pa13        9H-Fluoren-9-one              2.353
Pa14        C4-naphthalene                2.382
Pa15        phenanthrene                  2.442
Pa16        anthracene                    2.461
Pa17        C5-naphthalene                2.470
Pa18        C5-naphthalene                2.643
              (or C2-azulene)
Pa19        C5:1-naphtalene               1.649
Pa20        C5-naphtalene                 1.739
Pa21        C5:1-naphtalene               1.767
Pa22        C5-naphtalene                 1.801
Pa23        C5-naphtalene                 1.850
Pa24        C5-naphtalene                 1.853
Pa25        C5-naphtalene                 1.928
Pa26        C5-naphtalene                 1.974
Pa27        C5-naphtalene                 1.987
Pa28        C4-naphtalene                 2.000
Pa29        C5:1-naphtalene               2.037
Pa30        C5:1-naphtalene               2.143
Pa31        C5:1-naphtalene               2.211
N1          pyridine                      0.396
N2          pyrrole                       0.402
N3          acetamide                     0.421
N4          C1-pyrrole                    0.501
N5          C1-pyrrole                    0.521
N6          C1-pyridine                   0.543
N7          benzonitrile                  0.749
N8          pyridinecarbonitrile          0.887
N9          C1-benzonitrile               1.011
N10         C1-benzonitrile               1.067
N1l         1,3-benzenedicarbonitrile     1.302
N12         picolinamide                  1.320
N13         1,3-bcnzenedicarbonitrile     1.322
N14         isoquinoline                  1.334
N15         indole                        1.457
N16         phthalamic acid               1.457
N17         1,3-benzenedicarbonitrile     1.463
N18         C1-indole                     1.664
N19         C1-pthalimide                 1.719
N20         cyanobenzoic acid             1.827
N21         phenylpyridine                1.833
N22         chitin-derived compound       1.883
N23         diketodipyrrole               2.280
N24         diketopiperazine compound     2.646
Ps1         acetic acid                   0.203
Ps2         furanone compound             0.434
Ps3         3/2-furaldehyde               0.483
Ps4         acetylfuran                   0.613
Ps5         5-methyl-2-furaldehyde        0.686
Ps6         4-hydroxy-5,6-                0.755
              dihydro-(2H)-pyranone
Ps7         dianhydrorhamnose             0.861
Ps8         cyclopentenedione             0.869
              compound
Ps9         levoglucosenone               0.989
Ps10        3-hydroxy-2-methyl-           1.046
              4H-pyran-4-one
Ps11        dianhydro-[alpha],            1.263
              D-glucopyranose
Ps12        dibenzofuran                  1.953
Ps13        levoglucosan                  2.123
U1          alpha phellandrene            0.827
U2          U2 (possibly                  0.710
              methylcyclohexane)
U3          U3                            1.871
U4          U4                            2.186
U5          U5                            2.247
U6          U6                            3.152


Acknowledgements

We thank Antonio Martinez-Cortizas for the discussion on the statistical analysis. The contribution of M. Camps-Arbestain to this research was funded by MAF and NZAGRC. The authors also thank the anonymous reviewers for their constructive comments on the manuscript.

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Manuel Suarez-Abelenda (A,E), Joeri Kaal (B), Marta Camps-Arbestain (C), Heike Knicker (D), and Felipe Macias (A)

(A) Departamento de Edafoloxia e Quimica Agricola, Facultade de Bioloxi'a, Universidade de Santiago de Compostela, 15782- Santiago de Compostela, Spain.

(B) Instituto de Ciencias del Patrimonio (Incipit), Consejo Superior de Investigaciones Cientificas (CSIC), Rua San Roque 2, 15704 Santiago de Compostela, Spain.

(C) Institute of Natural Resources, Private Bag 11222, Massey University, Palmerston North 4442, New Zealand.

(D) Instituto de Recursos Naturales y Agrobiologia de Sevilla (IRNAS-CSIC), Adva. Reina Mercedes 10, 41012 Sevilla, Spain.

(E) Corresponding author. Email: manuel.suarez@usc.es

Table 1. General information of samples studied from soil PRD-4
[OC.sub.per], Permanganate-oxidisable organic C; [OC.sub.dichro],
dichromate-oxidisable

Sample                       S1                    S2

Depth                     5-10 cm              95-100 cm
Conventional           104.3 [+ or -]       4090 [+ or -] 30
  [sup.14]C               0.4 pMC
  age BP                 (present)
Radiocarbon
  sample code             Ua-34719           [beta]-299230
C (mg [g.sup.-1]
  soil)              62.3 [+ or -] 0.6     36.7 [+ or -] 0.3
[OC.sub.per] (mg
  [g.sup.-1] soil)    2.3 [+ or -] 0.1      1.3 [+ or -] 0.1
[OC.sub.dichro]
  (mg [g.sup.-1]
  soil)              27.5 [+ or -] 0.6     12.3 [+ or -] 0.3
C/N (atomic)                15.9                  24.4
pH-[H.sub.2]0               4.6                   5.0
Charcoal >2 mm
  (mg [g.sup-1]
  soil)                     0.03                  1.97

Sample                       S3

Depth                    190-195 cm
Conventional          9760 [+ or -] 50
  [sup.14]C
  age BP
Radiocarbon
  sample code          [beta]-240963
C (mg [g.sup.-1]
  soil)              42.9 [+ or -] 0.4
[OC.sub.per] (mg
  [g.sup.-1] soil)    1.7 [+ or -] 0.1
[OC.sub.dichro]
  (mg [g.sup.-1]
  soil)              14.3 [+ or -] 0.1
C/N (atomic)                23.9
pH-[H.sub.2]0               5.2
Charcoal >2 mm
  (mg [g.sup-1]
  soil)                     0.07

Table 2. Relative contributions of pyrolysis product groups
and benzene/alkylbenzenes and PAH/alkyl-PAHs ratios of total
quantified peak area (%TQPA)

Tot. Total; >[C.sub.18], long/chain n-alkenes/alkanes;
[C.sub.18]/[C.sub.10], short/cham H-alkenes/alkanes; OA,
other aliphatic compounds (predominantly branched alkenes);
Ph, phenols; TL, total lignin markers; MAH monocyclic
aromatic hydrocarbons; AB, alkyl/benzenes; PAH, polycyclic
aromatic hydrocarbons (intact terpene/like plant biomass,
ITPB; black/carbon/derived, BC); N, nitrogen compounds; BN,
BC/derived N compounds; Polysacchs, polysaccharides (well
preserved, WP; degraded, Dg); U, unidentified compounds; B/
AB, benzene/alkyl-benzenes ratio; P/AP, total PAH/alkylated
PAH ratio Second row: relative proportions within main group
(n-alkanes/enes, MAHs, PAHs, N compounds and
polysaccharides). Main groups are in bold

                      n-Alkenes

                                    [C.sub.18] -
               Tot.   >[C.sub.8]    [C.sub.10]

NO-1   %TQPA   1.7#       0.9           0.8
         %               51.4           48.6
MN-1   %TQPA   1.6#       0.5           1.1
         %               33.6           66.4
CR-1   %TQPA   9.3#       3.0           6.3
         %               32.2           67.8
NO-2   %TQPA   1.8#       0.6           1.2
         %               34.2           65.8
MN-2   %TQPA   2.5#       0.7           1.9
         %               26.0           74.0
CR-2   %TQPA   4.6#       1.6           3.0
         %               35.3           64.7
NO-3   %TQPA   0.9#       0.3           0.5
         %               36.6           63.4
MN-3   %TQPA   2.7#       0.8           1.9
         %               30.2           69.8
CR-3   %TQPA   3.5#       1.2           2.3
         %               35.0           65.0

               n-Alkanes

                             [C.sub.18] -
       Tot.    >[C.sub.8]    [C.sub.10]

NO-1   1.9#        0.8           1.1
                  40.1           59.9
MN-1   2.0#        0.3           1.7
                  13.4           86.6
CR-1   10.2#       3.3           6.9
                  32.2           67.8
NO-2   1.8#        0.6           1.2
                  34.1           65.9
MN-2   3.2#        0.5           2.7
                  16.7           83.3
CR-2   2.5#        0.9           1.5
                  37.7           62.3
NO-3   1.0#        0.5           0.5
                  47.3           52.7
MN-3   2.9#        1.2           1.7
                  42.0           58.0
CR-3   2.6#        1.1           1.5
                  41.1           58.9

                        [C.sub.2]-          Tot.
       OA (A)    Ph         Ph        TL     MAH     AB     Tot

NO-1    1.4     16.6#      1.8       7.4#   16.4#   6.6    10.6#
                                                    40.0
MN-1    4.1     14.1#      1.9       3.0#   30.0#   13.4   12.3#
                                                    44.7
CR-1    14.6    10.9#      1.4       1.0#   26.3#   4.6    4.8#
                                                    17.5
NO-2    1.7     13.7#      0.8       1.8#   20.4#   4.9    5.7#
                                                    23.8
MN-2    17.4    11.6#      0.9       1.4#   20.4#   3.6    3.3#
                                                    17.5
CR-2    7.0     4.8#       0.5       0.8#   16.9#   2.0    3.4#
                                                    11.6
NO-3    1.5     6.6#       0.0       0.7#   10.3#   2.5    1.4#
                                                    24.0
MN-3    9.2     7.7#       0.0       1.2#   17.5#   4.1    2.4#
                                                    23.2
CR-3    10.7    5.0#       0.0       2.2#   22.3#   2.7    2.5#
                                                    11.9

                           N              Polysacchs
       PAH
       ITPB    BC    Tot.     BN    Tot.     WP     Dg

NO-1   10.1   0.5    10.7#   3.6    30.4#   17.8   12.4
       95.6   4.4            33.2           58.4   40.7
MN-1   11.4   1.0    11.7#   4.8    18.4#   10.4   7.7
       92.2   7.8            40.7           56.6   41.7
CR-1   2.2    2.6    15.8#   1.4    6.4#    0.8    5.4
       45.7   54.3           47.0           12.4   84.7
NO-2   4.2    1.5    20.9#   11.9   31.5#   4.0    27.1
       73.0   27.0           57.1           12.6   86.3
MN-2   2.2    1.2    22.2#   13.9   17.4#   3.3    13.6
       65.0   35.0           62.6           18.7   77.9
CR-2   0.9    2.5    32.2#   23.8   27.6#   15.7   11.5
       25.3   74.7           74.1           57.0   41.8
NO-3   0.9    0.5    15.0#   9.0    62.2#   2.7    59.2
       65.0   35.0           60.2           4.4    95.2
MN-3   1.4    1.0    38.3#   28.9   17.7#   1.1    16.0
       57.9   42.1           75.5           6.5    90.7
CR-3   0.7    1.7    22.1#   16.5   28.8#   16.5   11.4
       30.0   70.0           74.8           57.5   39.6

           U           B/AB         P/AP

NO-1      2.5          0.2          0.02

MN-1      2.7          0.2          0.04

CR-1      0.7          1.2          0.60

NO-2      0.7          0.9          0.21

MN-2      0.5          1.3          0.26

CR-2      0.2          3.9          1 31

NO-3      0.3          1.3          0 35

MN-3      0.4          0.8          0.34

CR-3      0.3          2.7          0.89

Aliphatic compound with mass 83+280 (likely associated to fresh OM)
was not added because it is not indicative of the charring effect.

Note: Main groups are indicated with #.

Table 3. Chemical shift region distribution
(relative proportions, %) obtained from
solid-state [sup.13]C NMR

        Alkyl C     N-alkyl C,    O-alkyl C   C-H Aromatic C
       (0-45 ppm)   methoxyl C      (60-          (110-
                    (45-60 ppm)    110ppm)       140 ppm)

NO-1       29            9           29             14
MN-1       34            8           26             11
CR-1       29            4           22             19
NO-2       23            5           26             19
CR-2       10            2           16             42
NO-3       19            5           11             31

                          Carboxyl
       COR Aromatic C    C, amide C       Ketone C,
          (140 -           amide C       aldehyde C
          160 ppm)      (160-220 ppm)   (220-245 ppm)

NO-1         3               15               1
MN-1         2               17               1
CR-1         6               20               0
NO-2         1               21               4
CR-2         3               23               4
NO-3         5               26               3
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Author:Suarez-Abelenda, Manuel; Kaal, Joeri; Camps-Arbestain, Marta; Knicker, Heike; Macias, Felipe
Publication:Soil Research
Article Type:Report
Geographic Code:4EUSP
Date:Mar 1, 2014
Words:12382
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