Physical protection of lignin by organic matter and clay minerals from chemical oxidation

Organic Geochemistry 58 (2013) 1–12

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Physical protection of lignin by organic matter and clay minerals from chemical oxidation Joyce S. Clemente, Myrna J. Simpson ⇑ Environmental NMR Centre and Department of Chemistry, University of Toronto, Toronto, ON, Canada M1C 1A4

a r t i c l e

i n f o

Article history: Received 22 May 2012 Received in revised form 29 November 2012 Accepted 13 February 2013 Available online 26 February 2013

a b s t r a c t The role of organic matter (OM) concentration, structure and composition and how these relate to mineral protection is important for the understanding of long term soil OM dynamics. Various OM–clay complexes were constructed by sequential sorption of lignin and dodecanoic acid to montmorillonite. Humic acid–montmorillonite complexes were prepared at pH 4 and 7 to vary OM conformation prior to sorption. Results obtained with constructed OM–clay complexes were tested with isolated mineral fractions from two soils. Oxidation with an acidic NaClO2 solution was used to chemically oxidize lignin in the OM–clay complexes, sand-, silt- and clay-size soil fractions to test whether or not it can be protected from chemical attack. Gas chromatography–mass spectrometry was used to analyze lignin-derived phenols, cutin OH–acid (after CuO oxidation), fatty acid and n-alkanol concentrations and composition. We found that carbon content was not solely responsible for lignin stability against chemical oxidation. Lignin was protected from chemical oxidation through coating with dodecanoic acid and sorption of humic acid to clay minerals in a stretched conformation at pH 7. Therefore, interactions between OM constituents as well as OM conformation are important factors that protect lignin from chemical oxidation. Lignin-derived phenol dimers in the Grassland-Forest Transition soil fractions were protected from chemical oxidation to a greater extent compared to those in Grassland soil fractions. Therefore, although lignin was protected from degradation through mineral association, the extent of this protection was also related to OM content and the specific stability of lignin components. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The preservation of soil organic matter (OM) is important for maintaining soil quality and productivity (Janzen et al., 1998). The level of OM decomposition is higher in sand-size fractions which has led to the hypothesis that OM in fine sized soil fractions is protected from biodegradation through associations with mineral surfaces (Baldock et al., 1992; Guggenberger et al., 1995; Christensen, 2001; Quideau et al., 2001; Six et al., 2002). Furthermore, radiocarbon data (14C) used to estimate OM age coupled with stable isotope (d13C) turnover studies, suggest that silt- and clay-size fractions contain older and more slowly degraded OM (von Lutzow et al., 2007) and long term preservation of OM is attributed to its association with fine soil fractions (Christensen, 2001; Six et al., 2002; Kaiser and Guggenberger, 2003; Mikutta et al., 2007). Of particular interest is the stabilization of OM in clay-size fractions, because this fraction may contain as much as 50–75% of total soil OM (Christensen, 2001). However, clay surfaces have a finite amount of interaction sites, which can be saturated at high organic carbon concentrations (Six et al., 2002). Mikutta et al. (2006b) reported that OM was protected from degra⇑ Corresponding author. Tel.: +1 416 287 7234; fax: +1 416 287 7279. E-mail address: [email protected] (M.J. Simpson). 0146-6380/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.orggeochem.2013.02.007

dation, through association with clay minerals. Sorption studies also suggest that clay mineralogy, structure and composition influenced the structural composition of OM sorbed to clay surfaces (Asselman and Garnier, 2000; Feng et al., 2005; Simpson et al., 2006; Ghosh et al., 2009). It is therefore important to understand factors that may contribute to the stability of OM associated with clay-size fractions to better manage OM input and organic carbon sequestration in soils (Christensen, 2001; Six et al., 2002). OM associated with clay-size fractions may be stabilized through a number of physical and chemical interactions (Christensen, 2001; Six et al., 2002; Kögel-Knabner et al., 2008). One of which is the formation of microaggregates, where clay particles are coated with OM, which then subsequently limits access of degrading enzymes resulting in OM protection (Six, 2004; Chenu and Plante, 2006). Sorptive interactions between OM and clay mineral surfaces, which are governed by van der Waals interactions, ligand exchange, divalent cation bridging, electrostatic or hydrophobic bonding (Feng et al., 2005; Mikutta et al., 2007), also contribute to OM stabilization by mineral surfaces. The dominant mode of bonding is determined by solution properties (pH, ionic strength, presence of cations), valence of cations in solution, presence of competing ions (such as polyphosphate), properties of minerals and composition of the OM sorbate (Asselman and Garnier, 2000; Chi and Amy, 2004; Feng et al., 2005; Mikutta et al., 2007;

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J.S. Clemente, M.J. Simpson / Organic Geochemistry 58 (2013) 1–12

Ghosh et al., 2009). For example, ligand exchange was found to contribute to the sorption of peat humic acid to montmorillonite clay at weakly acidic pH values (Feng et al., 2005). OM was also found to sorb to goethite clay mainly through ligand exchange, while Ca2+ mediated cation bridging was found to be the dominant mechanism in OM sorption to vermiculite clay (Mikutta et al., 2007; Ghosh et al., 2009). The difference in strength and relative contribution of these sorption mechanisms was found to also play a role in the desorption of OM from clay and subsequent biodegradability (Mikutta et al., 2007). The fate of lignin in soil environments is also hypothesized to be regulated by interactions with mineral surfaces (Heim and Schmidt, 2007a,b). Model sorption experiments have observed higher concentrations of aromatic structures sorbed to montmorillonite as compared to kaolinite (Feng et al., 2005) and suggests that lignin stabilization in soil may also be tied to interactions with clay minerals. Recent studies have reported that lignin-derived phenols are at a more advanced oxidation stage in clay-size fractions as compared to those in sand- and silt-size fractions (Kiem and Kögel-Knabner, 2003; Thevenot et al., 2010; Clemente et al., 2011). Recent studies also suggest that lignin phenols may be sequestered in silt-size fractions and may be older and less oxidized compared to those in clay-size fractions (Heim and Schmidt, 2007a). In contrast, Feng et al. (2008) reported accelerated lignin oxidation with 14 months of soil warming. The authors hypothesized that the soil type (sandy loam) provided little physical protection from enhanced microbial activity that was observed using phospholipid fatty acid concentrations. Therefore, it is important to ascertain whether lignin in clay-size fractions is preferentially degraded by microbes in sites with high clay content, or if oxidized lignin becomes sorbed to clay minerals and subsequently leads to longer environmental persistence. It is also necessary to determine whether lignin associated with clay is protected from degradation and to characterize the factors that influence this protection because lignin associated with clay minerals represent an important part of stabilized OM (Thevenot et al., 2010). In this study, the factors that contribute to the stabilization of lignin on clay mineral surfaces were investigated using model OM compounds sorbed to montmorillonite and clay-size fractions isolated from soil. The influence of OM conformation was tested by creating humic acid–clay complexes at pH 4 and 7, since humic substances form coiled and compact aggregates at acidic pH and become stretched and disaggregated as pH increases (Chien and Bleam, 1998; Avena and Wilkinson, 2002). These OM–mineral compounds were subjected to chemical oxidation to test whether or not lignin is physically protected from enhanced chemical oxidation. We also examine the role of other OM compounds in the protection of lignin, as this was identified in our previous study as an important consideration of OM stabilization in soils (Clemente et al., 2011). The objectives of this study are to (1) determine whether the presence of other OM compounds can protect lignin from chemical oxidation, (2) determine the role of OM concentration in protection of lignin from chemical oxidation, (3) determine whether the sorption mechanism and OM conformation influences lignin protection at pH 4 and pH 7 and (4) test findings from model OM–clay complexes by examining lignin protection from chemical oxidation in the sand-, silt-, and clay-size fractions of two soils, which have similar clay mineralogy.

2. Materials and methods 2.1. Preparation of organic matter–clay complexes We prepared four types of model OM–clay complexes: lignin– clay, lignin–clay coated with dodecanoic acid and peat humic acid

sorbed to clay at pH 4 and 7. Control OM–clay complexes served as the reference point for ascertaining the relative changes to OM composition with chemical oxidation (see Section 2.5 for more information on data analysis and Fig. S1; Supplementary material). Sodium-rich montmorillonite (SWy-2) was purchased from the Source Clay Repository (Clay Minerals Society; Purdue University). The clay was suspended in a 3 mM NaCl + 2 mM CaCl2 solution at a 1:500 weight ratio to maintain the concentrations of Na+ and Ca2+, which are the major exchangeable cations associated with this clay mineral. Dissolved lignin (alkali lignin; Sigma–Aldrich) was added to the suspension (pH = 7), using the following concentrations: 0.4, 4, 20 and 100 g lignin/100 g clay to reflect the varying amounts of OM found in soils. Preliminary experiments found that vanillyl monomers were the main lignin phenols extracted using CuO oxidation from alkali lignin (Fig. S1; Supplementary material) and have been observed to be the most environmentally persistent lignin-derived phenols (Ertel and Hedges, 1984; Kiem and KögelKnabner, 2003; Bahri et al., 2006). The base soluble OM (humic acid) was isolated from the Pahokee Peat soil (International Humic Substances Society) as described by Salloum et al. (2001). This peat humic acid sample has been previously characterized by our laboratory using solid state and solution state nuclear magnetic resonance methods (Salloum et al., 2001; Feng et al., 2005) and the CuO oxidation products are shown in Fig. S1 (Supplementary material). Three humic acid–clay complexes were prepared in a similar manner as those with alkali lignin, with the following concentrations: 0.4, 4 and 20 g humic acid/100 g clay. The pH was adjusted to either 7.0 or 4.0 using HCl and NaOH, which changes the conformation of humic substances, coiled at pH 4 and stretched at pH 7 (Chien and Bleam, 1998; Avena and Wilkinson, 2002). The suspensions were placed on a shaker for 20 h, and then centrifuged at 4500 rpm for 1 h to isolate the OM–clay complexes. Complexes were then washed 10 times with the 3 mM NaCl + 2 mM CaCl2 salt solution to isolate the OM–clay complexes and avoid disturbing cation mediated associations. The complexes were freeze dried and ground to pass through a 106 lm sieve to reduce the contributions of any large aggregates. To create the lignin–clay–dodecanoic acid complexes, 1 g of the lignin–clay complexes (described as loadings 2, 3 and 4, which correspond to 4, 20 and 100 g lignin/ g clay, respectively) were mixed in acetone with 200 mg dodecanoic acid overnight on a shaker. Preliminary tests showed that the alkali lignin chosen for this study was soluble in water, but not in acetone. Therefore, extraction of lignin from the lignin–clay complexes was minimized by using acetone to dissolve the dodecanoic acid. This was verified by CuO oxidation followed by gas chromatography–mass spectrometry (GC–MS) analysis of the acetone supernatant which did not detect any lignin-derived phenols (data not shown). The OM–clay complexes were collected by centrifugation at 9500 rpm, washed twice with 30 ml acetone, then five times with the 3 mM NaCl + 2 mM CaCl2 salt solution. This was dried in the fume hood to remove residual acetone, then freeze dried and ground to pass through a 106 lm sieve. 2.2. Soil sampling and density fractionation Two samples from the Alberta Prairie Ecozone were collected: Southern Grassland (SG; collected near Lethbridge, Alberta) and Grassland-Forest Transition (GFT; collected near Tofield, Alberta) soils. Detailed descriptions of the soils and sampling areas can be found in Dudas and Pawluk (1969) and Janzen et al. (1998) and characteristics of the sand-, silt- and clay-size fractions are described by Clemente et al. (2011). These soils contain high amounts of montmorillonite and illite clays as well as chlorite and kaolinite but to a lesser extent (Bentley, 1979). Overlying vegetation in the SG soil site is dominated by Western Wheatgrass, while the GFT soil site is dominated by both grasses and stands of Quaking Aspen

J.S. Clemente, M.J. Simpson / Organic Geochemistry 58 (2013) 1–12

(Otto and Simpson, 2006a). Previous studies in our laboratory investigated these soils to characterize the relationships between climate and the composition of OM at the molecular level (Otto and Simpson, 2005, 2006a,b, 2007; Feng and Simpson, 2007; Clemente et al., 2011). Our previous studies indicated that these soils contain different amounts of lignin-derived phenols, cutin-derived OH–acids and other compounds which implied varying levels of OM degradation and inputs (Otto et al., 2005; Otto and Simpson, 2006a,b). After collection, these soils were air dried, passed through a 2 mm mesh sieve and then stored at room temperature in glass containers. Sand-, silt- and clay-size fractions were isolated using density and size fractionation techniques (Gregorich and Beare, 2008; Clemente et al., 2011). All fractions were freeze dried, then ground and stored at room temperature prior to analysis. Sample yields (which included light fraction, not used in this study) revealed that recovery was 95% for each sample but this does not seem to reflect any selective OM compound loss because we observed the same constituents but in varying distribution as previous studies, which analyzed these same soil samples (Otto and Simpson, 2005, 2006a,b). 2.3. Chemical oxidation of soil density fractions and organic matter– clay complexes Approximately 0.5 g of OM–clay complex, peat soil, silt- or claysize fraction and 1 g of sand-size fraction (n = 3) were suspended in a 20 ml salt solution containing 0.5 M phosphate buffer, which maintains the pH at 4.5, 3 mM NaCl and 2 mM CaCl2. OM–clay complexes and soil physical fractions (n = 3) were oxidized by dissolving 1 g of NaClO2 in the salt solution (resulting in a 5% weight concentration). NaClO2 concentrations were determined using preliminary experiments with high lignin concentrations to ensure that enough chemical oxidant was added for the complete chemical oxidation of lignin. Samples that were suspended in salt solution without NaClO2 were compared to chemically oxidized samples, such that changes in lignin oxidation and OM composition due to chemical oxidation can be determined. Both chemically oxidized and non-oxidized samples were then centrifuged for 30 min at 9500 rpm. The samples were suspended and washed with the 3 mM NaCl + 2 mM CaCl2 solution five times, freeze dried and ground prior to solvent extraction and CuO oxidation. 2.4. Carbon content, solvent extraction and lignin-derived phenol analysis OM–clay complexes, peat humic acid, peat soil and soil fractions were analyzed for total carbon using the LECO combustion method at the University of Guelph (Ontario, Canada). Using the method of Bundy and Bremner (1972), Otto and Simpson (2006a) did not detect inorganic carbon in the surface horizons of the soils: therefore, total carbon represents organic carbon content. Montmorillonite clay was also analyzed for carbon and the value (0.14% carbon by weight) was subtracted from the OM–clay complexes to determine sorbed organic carbon concentrations. Samples that were chemically oxidized (n = 3) and samples that did not receive NaClO2 were sequentially extracted with methanol (Fisher Scientific), dichloromethane:methanol (Fisher Scientific; 1:1 v:v), and dichloromethane to extract free lipids (Otto and Simpson, 2005). Only one sample that did not receive NaClO2, for each complex and soil fraction, was used because preliminary experiments (using lignin–clay complexes and soil fractions) indicated that the standard error in oxidized samples were greater compared to samples without NaClO2. The combined solvent extracts were then filtered through GF/A and GF/F glass microfibre filters, concentrated by rotary evaporation, and dried under a

3

stream of nitrogen gas (Praxair, Toronto, ON) in 2 ml vials. Lignin-derived phenols were extracted using CuO oxidation following the method of Hedges and Ertel (1982), as modified by Otto and Simpson (2006a). Cutin-derived hydroxy acids (OH–acids) can also be extracted with CuO oxidation (Goni and Hedges, 1990; Filley et al., 2008; Mendez-Millan et al., 2010). After solvent extraction, the OM–clay complexes and soil residues were air dried and then extracted with copper(II) oxide and ammonium iron(II) sulfate hexahydrate [Fe(NH4)2(SO4)26H2O] with 2 M NaOH in Teflon lined bombs. The bombs were flushed with nitrogen and incubated at 170 °C for 2.5 h. The supernatants were acidified to pH 1 using 6 M HCl (Caledon Laboratories, ON) and kept for 1 h at room temperature, in the dark to prevent cinnamic acid polymerization. After centrifugation at 2700 rpm for 30 min, the supernatant was extracted with diethyl ether (Fisher Scientific); the extracts were concentrated, transferred to 2 ml glass vials and then dried under a stream of nitrogen gas. The CuO oxidation residues were re-dissolved in dichloromethane prior to derivatization. Both solvent extracts and CuO extracts were derivatized by adding N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA; Sigma–Aldrich, Columbus, GA) and pyridine, followed by heating at 70 °C for 1 h. After cooling, the samples were analyzed using GC–MS. To do this, 3 ll of sample was injected using an Agilent 7683 autosampler (Agilent Technologies, Santa Clara, CA) in splitless mode. The sample was then eluted from the Agilent 6890N GC, which had an HP-5MS fused silica capillary column (30 m  0.25 mm i.d.  0.25 lm film thickness) by using the following temperature gradient: hold at 65 °C for 2 min, followed by a temperature increase from 65 °C to 300 °C at a rate of 6 °C per min and a final isothermal hold at 300 °C for 20 min. The Agilent 5973N mass spectrometer was operated at 70 eV in the electron impact mode. Data were acquired and processed using the Agilent Chemstation G1701DA v. D software. Compounds were identified by comparison with mass spectra from commercial libraries (Wiley, NIST) and authentic standards. Lauric acid (Sigma–Aldrich) was used as an external standard to estimate the relative amounts of organic acids and n-alkanol in the solvent extracts. Relative amounts of extractable lignin phenols were estimated by using vanillic acid (Sigma–Aldrich) as an external standard, while relative amounts of OH–acids were estimated using lauric acid as an external standard. Eight main lignin-derived phenol monomers were identified and quantified according to Hedges and Ertel (1982) and Otto and Simpson (2006a) which included vanillyl (vanillin, acetovanillone, vanillic acid), syringyl (syringaldehyde, acetosyringone, syringic acid) and coumaryl (coumaric acid, ferulic acid) groups. The relative contributions (%) of these three lignin monomer groups were calculated to determine changes in their distribution in the various samples. The acid/aldehyde ratios of vanillyl (Ad/Alv) and syringyl (Ad/Als) groups were calculated as these reflect the oxidation state of lignin-derived phenols, where higher values indicate that lignin is more oxidized (Hedges et al., 1988). Ligninderived phenol dimers were also identified according to Goni and Hedges (1992) and Otto and Simpson (2006a): these comprised 2-syringylsyringic acid, 2-syringylsyringaldehyde, 2-vanillylsyringic acid, dehydrodivanillic acid, dehydrovanillinvanillic acid, dehydrovanillinacetovanillone, dehydrodivanillin and dehydroacetovanillonevanillic acid. The concentrations of lignin-derived phenol dimers were divided by the concentrations of ligninderived phenol monomers (dimers/monomers). Cutin-derived OH–acids were also observed in the CuO oxidation extracts and these were identified according to Goni and Hedges (1990), Filley et al. (2008), and Mendez-Millan et al. (2010): these comprised 16-hydroxyhexadecanoic acid, 12-hydroxy-octadecanedioic acid, 9,10-dihydroxyhexadecanoic acid and 9,10,18-trihydroxyoctadecanoic acid. Similar to lignin phenol dimers, the relative

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J.S. Clemente, M.J. Simpson / Organic Geochemistry 58 (2013) 1–12

between oxidized samples and samples that did not receive NaClO2 (controls): change = ([oxidized sample-control]/control)  100. Here, a positive change indicates an increase after oxidation and a negative value indicates a decrease after oxidation. Multivariate comparisons were performed on chemical groups identified in solvent extracts and CuO oxidation extracts (n = 3) using SPSS v. 19.0. Analysis of variance followed by Tukey Honestly Significant Difference (HSD) were used to determine whether differences between mean changes were significant (a = 0.05). A 2-tailed, independent sample t-test was used to determine significant differences (a = 0.05) between lignin–clay and lignin–dodecanoic acid–clay complex, complexes prepared by humic acid sorption to clay at pH 4 and sorption at pH 7, and SG and GFT soils.

Table 1 Characteristics of lignin–clay complexes and lignin–clay complexes coated with dodecanoic acid prior to chemical oxidation. Lignin loadings (1 through 4) correspond to lignin concentrations of (1) 0.4, (2) 4, (3) 20, (4) 100 g lignin/100 g clay. Lignin loading 1

2

3

4

0.2 529 1.0

1.0 811 1.3

2.5 1989 0.6

4.1 2551 0.5

Lignin–clay complexes coated with dodecanoic acid C content (%) 1.6 Lignin phenols (lg/gsample) 727 Ad/Alv 0.8 Dodecanoic acid (lg/gsample) 260

3.1 1408 0.6 485

4.4 1483 0.5 367

Lignin–clay complexes C content (%) Lignin phenols (lg/gsample) Ad/Alv

3. Results and discussion amounts of OH–acids compared to lignin phenol monomers were calculated (OH–acids/monomers). The dimers/monomers and OH–acids/monomers ratios were used to determine the stability of lignin phenol monomers relative to lignin phenol dimers and cutin-derived OH–acids against oxidation. Characteristics of lignin– clay, lignin–clay coated with dodecanoic acid, humic acid–clay and peat soil and soil size fractions before chemical oxidation are listed in Tables 1–3 respectively.

3.1. Composition of organic matter–clay complexes before chemical oxidation Higher concentrations of organic carbon and extractable lignin phenols in OM–clay complexes were observed with higher lignin loadings (Table 1). Similarly, organic carbon content and lignin phenol concentrations in humic acid–clay complexes increased with humic acid loadings at both pH 4 and 7 (Table 2). This trend is consistent with previous studies where the amount of material sorbed to clay increased with higher starting concentrations of OM in solution (Asselman and Garnier, 2000; Feng et al., 2005; Bayrak, 2006; Ghosh et al., 2009). Only vanillyl phenol monomers (vanillin, acetovanillone, vanillic acid) were detected in the CuO

2.5. Data analysis Changes in the various parameters, which resulted from NaClO2 oxidation, were expressed as percentages of the difference

Table 2 Carbon content, composition of extractable lipids, lignin-derived phenols, and cutin-derived OH–acid contributions in humic acid–clay complexes before chemical oxidation. Humic acid loadings (1 through 3) correspond to: (1) 0.4, (2) 4 and (3) 20 g peat humic acid/100 g clay at pH 4 and pH 7. Humic acid loading

Peat soil

pH 4

C content (%) C9–C28 fatty acid (lg/gsample) n-alkanols (lg/gsample) Lignin phenols (lg/gsample) Ad/Alv Ad/Als % Vanillyl % Syringyl % Coumaryl Dimers/monomers OH–acid/monomers

pH 7

1

2

3

1

2

3

0.1 9 3 10 1.2 bdl 100 bdl bdl 2.3E7 7.3

1.1 12 4 135 1.3 0.6 37 48 16 0.3 0.2

3.9 11 2 456 2.1 1.4 57 35 9 0.04 0.01

0.1 9 4 8 1.3 bdl 100 bdl bdl 3.5E7 5.5

1.1 31 2 175 1.7 0.3 64 27 9 0.1 0.1

3.5 22 2 370 1.7 0.9 59 34 7 0.01 0.01

45.7 408 36 2130 2.8 2.1 55 33 13 0.02 0.06

bdl = below detectable limits.

Table 3 Carbon content, lignin-derived phenol and cutin-derived OH–acid composition in Southern Grassland and Grassland-Forest Transition sand-, silt- and clay-size fractions prior to chemical oxidation. Soil mineral fraction Southern Grassland (SG)

C content (%) Lignin phenols (lg/gsample) Ad/Alv Ad/Als Vanillyl (%) Syringyl (%) Coumaryl (%) Dimers/monomers OH–acids/monomers

Grassland-Forest Transition (GFT)

Sand

Silt

Clay

Sand

Silt

Clay

0.9 26 0.9 0.7 40 38 21 0.2 0.1

2.2 18 1.3 1.0 41 34 25 0.2 0.2

4.1 10 2.6 1.5 41 35 24 0.1 0.3

1.4 29 0.8 0.6 48 33 19 0.2 0.1

2.7 19 1.0 0.8 50 32 18 0.1 0.20

5.5 8 1.9 1.4 49 28 23 0.1 0.4

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J.S. Clemente, M.J. Simpson / Organic Geochemistry 58 (2013) 1–12

acid–clay complexes (notably loadings 2 and 3) constructed at pH 7, had greater organic acid concentrations as compared to humic acid–clay complexes constructed at pH 4 (Table 2), which may be due to a higher proportion of fatty acids that ionize at pH 7 (Kanicky et al., 2000) and subsequently sorbed to clay through Ca2+ mediated cation bridges (Mikutta et al., 2007). At higher pH values, humic acid adopts a more stretched conformation (Chien and Bleam, 1998; Avena and Wilkinson, 2002), which may also enhance interactions between fatty acids and the clay mineral surface. 3.2. Chemical oxidation of lignin–clay and lignin–clay–dodecanoic acid complexes Coating lignin–clay complexes with dodecanoic acid, a compound that is not altered by the chemical oxidant (Hoigne and Bader, 1994), resulted in further protection of vanillyl phenols from chemical oxidation (Fig. 1). Extractable lignin phenols from lignin–clay–dodecanoic acid complex increased slightly at the highest loading (Fig. 1; loading 4). An increase in extractable lignin phenols is believed to occur when lignin is depolymerized, but only slightly oxidized (Said-Pullicino et al., 2007). Vanillyl concentrations decreased to a greater extent in lignin–clay compared to lignin–dodecanoic acid–clay complexes at similar lignin loadings, suggesting that lignin protection from oxidation through OM–OM

(a)

20 Lignin

y

Lignin and dodecanoic acid

0

-20

xy -40

x

-60

c

bc

b -80

a -100 2*

1

(b)

Change in vanillyl oxidation (% Ad/Alv)

oxidation extracts of lignin–clay and lignin–dodecanoic acid–clay complexes (Table 1) and is consistent with the lignin phenol monomer composition of alkali lignin measured in preliminary studies (Fig. S1; Supplementary material). For lignin–clay complexes, the higher Ad/Alv ratios at lower lignin loadings (Table 1) suggest preferential association of more oxidized lignin phenols on clay surfaces. Vanillyl monomers were also preferentially sorbed to montmorillonite in humic acid–clay complexes (Table 2). In both SG and GFT soils, higher Ad/Al ratios in the clay- compared to sandand silt-size fractions suggest greater oxidation of lignin in claysize fractions. These data are consistent with previous studies, which concluded that lignin in fine particle-size fractions are more oxidized (Guggenberger et al., 1994; Quideau et al., 2001; Heim and Schmidt, 2007a; Clemente et al., 2011). Lignin phenols, which consisted mainly of vanillyl monomers, were also isolated from peat soil (Table 2) and enrichment of vanillyl monomers in soils is attributed to the greater chemical recalcitrance of these lignin phenol structures (Hedges et al., 1988; Bahri et al., 2006). However, the affinity between oxidized lignin phenols, vanillyl monomers and clay mineral surfaces observed in the OM–clay complexes (Tables 1 and 2) and higher Ad/Al ratios in soil clay-size fractions also collectively suggest that there is preferential association between clay minerals and specific lignin phenol structures. Aliphatic compounds may have been associated with lignin– dodecanoic acid–clay and humic acid–clay complexes through different modes. Similar concentrations of dodecanoic acid were extracted from lignin–clay–dodecanoic acid complexes regardless of lignin concentrations (Table 1), which suggests that dodecanoic acid coated lignin–clay complexes to a similar extent regardless of the lignin loading used. These results indicate additional protection of OM through interactions between OM compounds such as lignin and dodecanoic acid, in addition to protection through OM–clay interactions. Similarly, proteins and n-alkanes are hypothesized to be protected from degradation through association with other soil OM structures (Simonart et al., 1967; Lichtfouse et al., 1998). On the other hand, both cutin-derived OH–acids and plant-derived organic acids are found in humic acid extracts from peat soil (Table 2). These compounds, along with other structures such as lignin phenols, are likely to simultaneously sorb to clay minerals during humic acid–clay complex formation. In the humic acid–clay complexes, the ratio of OH–acid to lignin phenol monomers decreased with increased humic acid loadings (Table 2) and indicates that OM sorbed to clay at higher humic acid concentrations consisted of a smaller proportion of cutin OH–acids. Peat soil, which has a high carbon content (Table 2) also had lower OH–acid to lignin phenol monomer ratios (Table 2), which is attributed to the slow rate of plant material degradation (Johnston et al., 1997). Previous studies, also found that aliphatic compounds (such as cutin) preferentially associate with clay mineral surfaces (Chi and Amy, 2004; Feng et al., 2005). These studies, together with our observations indicate that the smaller proportion of cutin OH–acids at higher humic acid loadings may present another mechanism for OM interaction with clay minerals. For example, at higher humic acid concentrations, aliphatic compounds on clay surfaces may enhance indirect association of other compounds (such as lignin and carbohydrates) with clay minerals through hydrophobic interactions between OM and previously sorbed aliphatic compounds. These interactions may be more favorable than direct interactions between compounds, such as lignin and carbohydrates, and clay mineral surfaces. Fatty acids with C9–C28, with a preference for even carbon number, as well as C18, C24 and C26 n-alkanol were also detected in the solvent extracts of the humic acid–clay complexes (Table 2). This even carbon preference is typical of plant-derived cuticular waxes, and is consistent with the plant origin of peat humic acid, while contributions from lipids with
3

4*

a

120 100

Lignin

80

Lignin and Dodecanoic acid

b

60 40 20 0 -20 -40

c c

-60 1

2*

3

4

Lignin loading Fig. 1. Lignin oxidation in lignin–clay and lignin–clay–dodecanoic acid complexes after chemical oxidation (a) changes in vanillyl monomer concentrations and (b) changes in vanillyl oxidation (Ad/Alv). Values plotted are changes (%) after chemical oxidation. Loading numbers (1–4) indicate higher lignin concentrations respectively prior to oxidation (Table 1). Positive values suggest an increase, while negative values suggest a decrease due to chemical oxidation. Error bars represent the standard error of the mean (n = 3 digests). Letters indicate significant difference between samples with different lignin loadings (Tukey HSD, a = 0.05), and () indicate significant difference between samples with and without dodecanoic acid (t-test, a = 0.05).

3.5

(a)

a

2.5

(% µg/g sample)

J.S. Clemente, M.J. Simpson / Organic Geochemistry 58 (2013) 1–12

(% mg/g sample)

6

1.5

150 110 70

pH 7

pH 4

b

30

b 0.5

-10

b -50 2

3

4

-0.5

y

-90

Lignin loading

a

interactions between lignin and dodecanoic acid may also have occurred (Fig. 1). Protection of lignin by dodecanoic acid may have limited access of water (and the oxidizing agent). In soils, hydrophobicity is hypothesized to limit the degradation of OM (Kleber, 2010). Long chain aliphatic compounds, such as dodecanoic acid, are also known to stabilize microaggregates (Dinel et al., 1991a,b), and protection of lignin within these aggregates may have also inhibited its chemical oxidation. At the highest lignin concentration (Fig. 1; loading 4), lignin phenol oxidation resulted in higher Ad/Alv ratios but less lignin was removed at this loading since vanillyl yields decreased to a lesser extent (Fig. 1; loading 4). However, at the lowest lignin concentration (Fig. 1; loading 1), there was a smaller increase in Ad/Alv ratios after chemical oxidation likely because the chemical oxidation products were no longer identified as distinct lignin phenols. These data suggest that greater concentrations of lignin on clay resulted in OM–OM interactions, which protected lignin from chemical oxidation. The concentration of dodecanoic acid that was extracted after chemical oxidation was also higher in lignin– clay–dodecanoic acid complexes with lower lignin concentrations (Fig. 2; loading 2). This pattern indicates that the stability of interactions between dodecanoic acid and the lignin–clay complex was also dependent on the amount of lignin sorbed to montmorillonite, which is disrupted as lignin is chemically oxidized (Fig. 2). This observation is consistent with the hypothesis that when OM sufficiently coats the active sites on clay minerals, the OM that is subsequently added sorbs to the previously formed OM layer through hydrophobic interactions (Kaiser and Guggenberger, 2000; Chi and Amy, 2004). Therefore, both the lower degradation of lignin phenols and lower concentrations of extractable dodecanoic acid at higher lignin loadings indicate that increased hydrophobic interactions and enhanced OM–OM interactions may further protect clayassociated OM.

3.3. Chemical oxidation of humic acid–clay complexes To investigate whether the composition and conformation of OM sorbed to montmorillonite plays a role in lignin protection from chemical oxidation; changes in lignin concentration, composition and extractable n-alkanol and fatty acids after chemical oxidation were measured for humic acid–montmorillonite complexes prepared at pH 4 and 7 (Figs. 3–5). Despite differences in the conformation of humic acid sorbed to clay minerals at pH 4 and 7,

a y

a

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2

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4500

Change in OH-acids/monomers (%)

Fig. 2. Increase (%) in extractable dodecanoic acid after chemical oxidation of lignin–clay–dodecanoic acid complexes. Values plotted are differences in % extracted before and after oxidation, and larger loading numbers (2–4) indicate higher lignin concentrations prior to oxidation (Table 1). Positive values suggest an increase in extracted dodecanoic acid as a consequence of lignin oxidation. Error bars represent the standard error of the mean (n = 3 digests). Different letters indicate significant difference in extractable dodecanoic acid among complexes with different lignin loadings (Tukey-HSD, a = 0.05).

x

x

3500

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ab xy

xy 1500 500 -500

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Humic acid loading Fig. 3. Changes in lignin concentrations in humic acid–clay complexes (created at pH 4 and 7) and peat soil after chemical oxidation: (a) decreased concentrations of lignin phenol monomers, and (b) increased OH–acid/monomer ratios. Values plotted are changes (%) after chemical oxidation, and larger loading numbers (1–3) indicate greater humic acid concentrations prior to oxidation (Table 2). Positive values suggest an increase, while negative values suggest a decrease due to oxidation. Error bars represent the standard error of the mean (n = 3 digests). Different letters indicate significant differences between complexes with different humic acid loadings (Tukey-HSD, a = 0.05), and () indicates significant difference between complexes created at pH 7 and pH 4 (t-test, a = 0.05).

there were similarities in the oxidation of lignin phenols in both sets of humic acid–clay complexes. With the exception of the pH 7 complex with the lowest humic acid concentration (Fig. 3; loading 1), lignin phenol concentrations decreased with chemical oxidation, which suggests lignin removal in the majority of humic acid–clay complexes (Fig. 3a). Increased OH–acid/monomer ratios (Fig. 3b), is also consistent with lignin removal and illustrates the resulting enrichment of cutin-derived OH–acids relative to lignin phenols with chemical oxidation. This enrichment is due to the 106–1011 times greater reactivity of phenols to chlorine dioxide, the main oxidizer produced by acidifying NaClO2 (Svenson et al., 2006), as compared to alcohols and fatty acids (Hoigne and Bader, 1994). Humic acid–clay complexes were created by simultaneous, competitive sorption of a complex mixture of OM compounds within the humic acid, which is in contrast to the layering of lignin–clay with dodecanoic acid. The simultaneous mode of sorption may have led to less efficient protection of lignin in the resulting humic acid–clay complexes (Fig. 3) compared to that in lignin– dodecanoic acid–clay complexes (Fig. 1). The number of sorption sites on clay surfaces are limited (Six et al., 2002; Kaiser and Guggenberger, 2003; Mikutta et al., 2007), and aliphatic compounds that preferentially sorb to montmorillonite (Feng et al., 2005; Ghosh et al., 2009) may out compete lignin for these sites.

7

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Change in syringyl oxidation (% Ad/Al s)

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Humic acid loading Fig. 4. Changes in lignin phenol composition in humic acid–clay complexes (created at pH 4 and 7) and peat soil after chemical oxidation: (a) changes in vanillyl contribution (vanillin + acetovanillone + vanillic acid) (b) decreased syringyl contribution (syringaldehyde + acetosyringone + syringic acid), (c) decreased coumaryl contribution (coumaric acid + ferulic acid), (d) changes in vanillyl oxidation (Ad/Alv), (e) changes in syringyl oxidation (Ad/Als), and (f) changes in dimers/monomers. Values plotted are changes (%) after chemical oxidation, and larger loading numbers (1–3) indicate greater humic acid concentrations prior to oxidation (Table 2). Positive values suggest an increase, while negative values suggest a decrease due to oxidation. Error bars represent the standard error of the mean (n = 3 digests). Different letters indicate significant difference in samples with different humic acid loadings (Tukey-HSD, a = 0.05), and () indicate significant difference between complexes created at pH 7 and pH 4 (t-test, a = 0.05).

Therefore, at high humic acid loadings, some lignin may have associated with clay mineral surfaces through interactions with OM, as suggested by lower OH–acid/lignin ratios at higher humic acid concentrations (Table 2). However, aromatic structures may not always interact with aliphatic structures within humic acids (Chien and Bleam, 1998) and the types of aliphatic components within peat humic acid vary considerably in terms of their affinity for clay surfaces (Feng et al., 2005). Thus, OM–OM interactions may also limit the degree of lignin protection. This hypothesis is further supported by similar levels of chemical oxidation of lignin phenols in peat soil and humic-acid clay complexes with higher humic acid concentrations (Fig. 3; loadings 2 and 3).

For humic acid–clay complexes created at pH 4 and 7, preferential oxidation of syringyl and coumaryl monomers resulted in higher proportions from vanillyl monomers after chemical oxidation (Fig. 4a). This trend is consistent with the reported stability of vanillyl compared to syringyl and coumaryl monomers (Ertel and Hedges, 1984; Bahri et al., 2006). As well, Ad/Alv ratios increased (Fig. 4d), while Ad/Als ratios decreased (Fig. 4e) after chemical oxidation. A previous study concluded that syringic acid and coumaric acid were more labile than ferulic acid, acetosyringone, syringaldehyde, and vanillyl monomers (Bahri et al., 2006). Therefore, syringic acid may not always accumulate relative to syringaldehyde, which leads to decreased Ad/Als. In contrast, vanillin is thought

J.S. Clemente, M.J. Simpson / Organic Geochemistry 58 (2013) 1–12

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Increased extractable organic acids (% µg/g sample)

(a)

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pH 7 1000

pH 4

a 600

ab b

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Humic acid loading Fig. 5. Increase in extractable (a) n-alkanol and (b) organic acids after chemical oxidation of humic acid–clay complexes (created at pH 4 and pH 7), and peat soil. Values plotted are changes (%) after chemical oxidation, and larger loading numbers (1–3) indicate greater humic acid concentrations prior to oxidation (Table 2). Error bars represent the standard error of the mean (n = 3 digests). Different letters indicate significant differences between samples with different peat humic acid loadings (Tukey-HSD, a = 0.05), and () indicate significant difference between complexes created at pH 7 and pH 4 (t-test, a = 0.05).

to be more labile than vanillic acid (Hedges et al., 1988; Bahri et al., 2006). The relative changes in lignin phenol composition (Fig. 4) therefore suggest that the relative recalcitrance of OM structures may also contribute to OM protection in OM–clay complexes. Despite the similarities in overall lignin oxidation for the complexes created at pH 4 and 7, pH controlled composition and conformation of OM sorbed to clay minerals, which resulted in noticeable differences in lignin protection. Lignin was removed to a lesser extent at the lowest humic acid concentration at pH 7 (Fig. 3; loading 1), which also had the lowest lignin phenol concentrations prior to oxidation (Tables 1 and 2). At these concentrations, lignin may have sorbed to high affinity sites, which inhibited OM degradation (Mikutta et al., 2007). Enhanced monolayer sorption on clay due to the stretched conformation of humic acids at pH 7 (Chien and Bleam, 1998; Avena and Wilkinson, 2002) may have also resulted in stronger OM–clay interactions, and lignin protection. Therefore, although aromatic structures (such as those in lignin phenols) may not be preferentially sorbed by montmorillonite (Asselman and Garnier, 2000), availability of high affinity sites on clay and monolayer sorption, are possible mechanisms that protected lignin phenols at the lowest humic acid concentrations from chemical oxidation (Fig. 3). In complexes with the highest carbon concentrations prior to oxidation (Table 2; loading 3), chemical oxidation resulted in increased Ad/Alv ratios, which was greatest in the humic–clay complex prepared at pH 4 compared to that prepared at pH 7 (Fig. 4d). This difference is likely because vanillin was less oxidized in the pH 7 complex, such that 30% of the vanillin was detected after oxidation, while only 2% of vanillin was detected in the

pH 4 complex after chemical oxidation (not shown). Since vanillic acid is a product of vanillin oxidation (Hedges et al., 1988), and changes in lignin phenol concentrations were similar for both pH values, these data suggest that vanillin in the pH 4 complex was more susceptible to oxidation. This hypothesis is further supported by the higher dimers/monomers ratio in the pH 7 compared to the pH 4 complex (Fig. 4f; humic acid loading 3), which resulted from higher residual dimer concentrations in the pH 7 complex. Although dimers/monomers ratios in soils increase through enrichment of more stable lignin dimers as lignin monomers are degraded (Goni and Hedges, 1992; Goni et al., 1993; Opsahl and Benner, 1995; Thevenot et al., 2010), the higher ratios in the pH 7 complex after oxidation may be attributed to better protection of lignin in the pH 7 humic acid–clay complex. After chemical oxidation, there was also a greater proportion of n-alkanol and organic acids extracted from complexes created at pH 4 and peat soil compared to complexes created at pH 7 (Fig. 5). These data further support the hypothesis that under the conditions used in this study, OM–clay complexes created at pH 7 were less susceptible to oxidation than those created at pH 4. The solution pH during the creation of OM–clay complexes may have influenced both OM conformation and the mechanisms responsible for OM–clay interactions (Piccolo et al., 1996; Chien and Bleam, 1998; Avena and Wilkinson, 2002; Feng et al., 2005; Baalousha et al., 2006; Mikutta et al., 2007). Solution pH is believed to influence the conformation of humic substances in that OM aggregation increases under acidic pH; while OM disaggregates under basic pH. These pH-induced OM conformations may also influence the structures exposed to solution and therefore the structures available to complex with clay, since a portion of the structures are protected within the OM aggregate. For example, associations between carboxyl groups, which are more likely to occur under acidic pH (Doan et al., 1997) may need to be disrupted before complexation between these structures and clay can occur. Solution pH may also influence the mechanisms responsible for OM–clay interactions because ligand exchange was observed only in clay–OM complexes created at pH 4 (Feng et al., 2005), and Ca2+mediated cation bridges were more important at neutral pH values (Mikutta et al., 2007). In this study, a PO3 4 buffer was used during oxidation, which may have weakened OM–clay sorption through ligand exchange (Kaiser and Guggenberger, 2000; Mikutta et al., 2006a), thereby increasing OM susceptibility to chemical oxidation (Mikutta et al., 2007). The stability of lignin against degradation in the pH 7 complex compared to the pH 4 complex may therefore be attributed to OM composition, and OM–clay interactions (such as ligand exchange) that are favored at pH 4. 3.4. Chemical oxidation of soil density fractions Using SG and GFT soil samples, protection of lignin from oxidation through association with sand-, silt-, and clay-size fractions in natural samples was investigated. Similar to OM–clay complexes, cutin-derived OH–acids were enriched as a result of lignin monomer chemical oxidation (Fig. 6). The depletion of syringyl and coumaryl monomers, which resulted in enrichment of vanillyl monomers (Fig. 7) is consistent with other observations that report enhanced stability of vanillyl monomers in the environment (Ertel and Hedges, 1984; Kiem and Kögel-Knabner, 2003; Bahri et al., 2006). Lignin removal and changes in lignin composition were also compared across the sand-, silt-, and clay-size fractions. A previous study suggests that lignin phenols were preferentially preserved in silt-size fractions (Heim and Schmidt, 2007a), likely through association with aggregates (Six, 2004). However, destruction of aggregates in the SG and GFT silt-size fractions (through grinding) may have resulted in less protection for OM as compared to lignin in clay-size fractions (Figs. 6 and 7). Decrease in lignin concentration

(a)

(% µg/g sample)

J.S. Clemente, M.J. Simpson / Organic Geochemistry 58 (2013) 1–12

0 -20 -40 -60

xy -80 -100

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y

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400

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GFT

b

300

200

c

100

0

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Soil mineral fraction Fig. 6. Decreased lignin concentrations in Southern Grassland (SG) and GrasslandForest Transition (GFT) soil mineral fractions after chemical oxidation: (a) decrease in lignin phenol monomers, and (b) increase in OH–acid/monomer ratios. Values plotted are differences (%) before and after chemical oxidation. Error bars represent the standard error of the mean (n = 3 digests). Different letters indicate significant differences between fractions (Tukey-HSD, a = 0.05), and () indicates significant difference between SG and GFT fractions (t-test, a = 0.05).

and changes in lignin phenol composition were smaller in clay-size compared to sand-, and silt-size fractions (Figs. 6 and 7), which suggests that OM associated with clay was protected from chemical oxidation. Such protection is consistent with the hypothesis that OM in finer fractions is older because they are physically protected from degradation (Baldock and Skjemstad, 2000; von Lutzow et al., 2007). Protection of OM in clay-size fractions observed in this study may be attributed to OM protection within smaller microaggregates, and primary physical or chemical associations between OM and clay. Microaggregates found in clay-sized fractions are believed to be stable against physical manipulations (Six, 2004; Chenu and Plante, 2006). Sorption of OM to clay also limits access of chemical reagents and enzymes to the reactive structures in OM (Christensen, 2001; Six et al., 2002; Mikutta et al., 2006b; von Lutzow et al., 2007). In contrast, chemical recalcitrance may be more important in protecting OM in sand-size fractions from oxidation because of the lower surface area and limited interactions between sand-size minerals and OM (Christensen, 2001). Lignin concentrations in the sand-size fractions decreased to a greater extent indicating that chemical recalcitrance may not be as effective as mineral association (Fig. 6) in protecting OM. This hypothesis is consistent with previously reported faster turnover of lignin in larger particle-size fractions (von Lutzow et al., 2007; Heim and Schmidt, 2007a). The extent of lignin oxidation in the SG particle-size fractions was also compared to those of GFT size fractions (Figs. 6 and 7) to determine whether differences in organic carbon content, and vegetation influenced lignin protection. The decrease in lignin phenol concentrations, enrichment of cutin-derived OH–acids, enrich-

9

ment of vanillyl monomers, and increases in Ad/Alv ratios after digestion (Figs. 6 and 7), in the sand- and silt-size fractions of SG was greater than those of GFT fractions, all of which suggest that lignin in the SG fractions were less protected. Because of the limited interactions between minerals and OM in sand-size fractions (Christensen, 2001), and disturbance of silt-size aggregates, differences in the oxidation of lignin in these fractions may be attributed to lignin phenol composition. This hypothesis is consistent with higher vanillyl monomer concentrations (Table 3), which are more stable than syringyl and coumaryl monomers (Ertel and Hedges, 1984; Kiem and Kögel-Knabner, 2003; Bahri et al., 2006); and higher vanillyl dimer concentrations (not shown), which are more stable than monomers (Goni and Hedges, 1992; Goni et al., 1993; Otto and Simpson, 2006a) in GFT fractions. Because OM in sandsize fractions are at an earlier stage of degradation, differences in lignin structures extracted from this fraction may be attributed to the overlying vegetation. The SG soil is dominated by Western Wheatgrass, while GFT soil is dominated by Quaking Aspen as well as grasses (Otto and Simpson, 2006a). The replacement of grass by trees has been observed to result in more recalcitrant soil OM (Liao et al., 2006). Susceptibility of grass-derived lignin to degradation is also consistent with its structure: it was observed to have greater concentrations of more labile coumaryl monomers (Nimz et al., 1981; Opsahl and Benner, 1995; Otto and Simpson, 2006a); and more ester linkages, which are easily decomposed (Nimz et al., 1981). These structural considerations are consistent with a previous study, in which Klason lignin extracted from grass was also more easily degraded by microorganisms, compared to those extracted from softwood and hardwood (Antai and Crawford, 1981). The oxidation levels of lignin in the clay-size fractions in both soils were similar, as were the increase in vanillyl, decrease in coumaryl monomer contributions, and increases in Ad/Al ratios (Figs. 6 and 7). These data suggest that overall, lignin monomers in the clay-size fractions of both soils were protected to the same extent. The clay mineral composition of both soils were dominated by montmorillonite and illite (Bentley, 1979). Therefore, the interactions between clay and OM that were responsible for protecting lignin from chemical oxidation may have been similar as well. However, syringyl monomer contributions decreased to a greater extent, while dimer contributions decreased to a lesser extent in the GFT compared to the SG clay-size fraction (Fig. 7b and f). These differences in the degradation levels of specific structures in SG and GFT, suggest that OM composition prior to oxidation may have also influenced lignin protection in the clay-size fractions, and is consistent with that observed in the humic acid–clay complex (Fig. 4) as well. For example, the greater resistance of lignin dimers in the GFT fractions (concentrations decreased by 16% in GFT, and 50% in SG clay-size fraction) may be attributed to the greater concentrations of dimers where aromatic rings are directly linked (e.g. 5–50 dimers) in GFT fractions, which is consistent with total soil dimer concentrations found by Otto and Simpson (2006a). These structures are thought to be more recalcitrant compared to when carbonyl or methyl groups link the aromatic structures (e.g. b,1-diketone dimers; a,1-monoketone dimers; a,5-monoketone dimers; and a,2methyl dimers (Goni and Hedges, 1992). The mechanism responsible for greater decrease of syringyl monomers in GFT clay-size fraction after NaClO2 oxidation on the other hand, is less clear, but may also be related to the structure of lignin in GFT vegetation. Therefore, although the overall degradation of lignin phenols in clay-size fractions was mainly limited by protection of OM through its interactions with clay minerals, inherent recalcitrance of lignin structures may have also influenced the relative degradability of specific structures. This is consistent with the hypothesis that OM structure influences its preservation in soil minerals (Kaiser and Guggenberger, 2000). It is also possible that lignin phenol dimers were unable to form strong interactions with SG clay, which

J.S. Clemente, M.J. Simpson / Organic Geochemistry 58 (2013) 1–12

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Soil mineral fraction Fig. 7. Changes in lignin phenol monomer composition of Southern Grassland (SG) and Grassland-Forest Transition (GFT) soils after chemical oxidation: (a) increased vanillyl contribution (vanillyl + acetovanillone + vanillic acid), (b) decreased syringyl contribution (syringaldehyde + acetosyringone + syringic acid), (c) decreased coumaryl contribution (coumaric acid + ferulic acid), (d) increased vanillyl oxidation, (e) increased syringyl oxidation, and (f) changes in dimers/monomers. Values plotted are changes (%) after chemical oxidation. Positive values suggest an increase in Ad/Al and dimers/monomers ratios, while negative values suggest decreases in values due to oxidation. Error bars represent the standard error of the mean (n = 3 digests). Different letters indicate significant difference between different fractions (Tukey-HSD, a = 0.05), and () indicate significant difference between SG and GFT soil fractions (t-test, a = 0.05).

made them more susceptible to oxidation. The total OM in this clay-size fraction contains higher concentrations of aliphatic structures (Clemente et al., 2011), which may compete with lignin for sorption sites, since montmorillonite sorbs high concentrations of aliphatic compounds (Feng et al., 2005).

4. Conclusions By examining OM–montmorillonite clay complexes and mineral fractions from two soils, we found that OM concentration and composition governed the protection of lignin from NaClO2 oxidation. Coating lignin–clay complexes with dodecanoic acid, which was resistant to NaClO2 oxidation, protected lignin from chemical oxidation. The stretched conformation of humic acids in humic acid–clay complexes created at pH 7, which may have promoted monolayer sorption of humic acids to montmorillonite, also

enhanced protection of lignin from chemical oxidation. Accordingly, interactions between OM structures may be another protection mechanism that should be considered. Current models emphasize the importance of interactions between OM and clay minerals and protection within microaggregates, as the main mechanisms responsible for OM protection in soils. Our results are consistent with these models in that overall, lignin in clay-size fractions of SG and GFT soils were protected against chemical oxidation to the same extent. However, OM composition also appears to play a role in lignin protection from chemical attack. This hypothesis is emphasized by the more advanced oxidation of lignin dimers in SG fractions, compared to GFT fractions; and the greater differences in the oxidation of lignin in sand- and clay-size fractions of SG soils compared to those of GFT soils. The role of OM structure and composition in its preservation should be considered further because it has been hypothesized that clay sorption sites may become saturated as soil carbon content increases. Therefore,

J.S. Clemente, M.J. Simpson / Organic Geochemistry 58 (2013) 1–12

as carbon concentrations increase, interactions between OM may become more important in determining preservation. It can also be envisioned that OM sorption to clay may become more stable with time, because of the additional protection provided by overlying OM. These OM–clay interactions may depend on the structures of both OM and clay minerals. Therefore, understanding how OM structures control their association with soil minerals and subsequent preservation, may help predict the resilience of protected OM against disturbance caused by changes in environmental conditions and land management. Future studies should examine the role of a variety of OM components to further test the observed trends from this study. Acknowledgements The authors thank Katherine Hills (funded through the Natural Sciences and Engineering Research Council Undergraduate Student Research Awards Program) for assistance with sample extractions. Funding for this project was provided by the Natural Sciences and Engineering Research Council Green Crop Network. J.S.C. also thanks the Ontario Government for support via an Ontario Graduate Scholarship. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.orggeochem. 2013.02.007. Associate Editor—Ian Bull References Antai, S.P., Crawford, D.L., 1981. Degradation of softwood, hardwood, and grass lignocellulose by two Streptomyces strains. Applied and Environment Microbiology 42, 378–380. Asselman, T., Garnier, G., 2000. Adsorption of model wood polymers and colloids on bentonites. Colloids and Surfaces A 168, 175–182. Avena, M.J., Wilkinson, K.J., 2002. Disaggregation kinetics of a peat humic acid: mechanism and pH effects. Environmental Science and Technology 36, 5100– 5105. Baalousha, M., Motelica-Heino, M., Le Coustumer, P., 2006. Conformation and size of humic substances: effects of major cation concentration and type, pH, salinity, and residence time. Colloids and Surfaces A 272, 48–55. Bahri, H., Dignac, M.F., Rumpel, C., Rasse, D.P., Chenu, C., Mariotti, A., 2006. Lignin turnover kinetics in an agricultural soil is monomer specific. Soil Biology & Biochemistry 38, 1977–1988. Baldock, J.A., Skjemstad, J.O., 2000. Role of the soil matrix and minerals in protecting natural organic materials against biological attack. Organic Geochemistry 31, 697–710. Baldock, J.A., Oades, J.M., Waters, A.G., Peng, X., Vassallo, A.M., Wilson, M.A., 1992. Aspects of the chemical structure of soil organic materials as revealed by solidstate 13C NMR spectroscopy. Biogeochemistry 16, 1–42. Bayrak, Y., 2006. Application of Langmuir isotherm to saturated fatty acid adsorption. Microporous and Mesoporous Materials 87, 203–206. Bentley, C.F., 1979. Photographs and descriptions of some Canadian soils: based on ‘‘The display of Canadian soils’’ at Eleventh Congress International Society of Soil Science, Edmonton, Canada, June 1978. University of Alberta. Bull, I.D., Nott, C.J., van Bergen, P.F., Poulton, P.R., Evershed, R.P., 2000a. Organic geochemical studies of soils from the Rothamsted Classical Experiments – VI. The occurrence and source of organic acids in an experimental grassland soil. Soil Biology & Biochemistry 32, 1367–1376. Bull, I.D., van Bergen, P.F., Nott, C.J., Poulton, P.R., Evershed, R.P., 2000b. Organic geochemical studies of soils from the Rothamsted classical experiments – V. The fate of lipids in different long-term experiments. Organic Geochemistry 31, 389–408. Bundy, L.G., Bremner, J.M., 1972. Simple titrimetric method for determination of inorganic carbon in soils. Soil Science Society of America Proceedings 36, 273– 275. Chenu, C., Plante, A.F., 2006. Clay-sized organo-mineral complexes in a cultivation chronosequence: revisiting the concept of the ‘primary organo-mineral complex’. European Journal of Soil Science 57, 596–607. Chi, F.-H., Amy, G.L., 2004. Kinetic study on the sorption of dissolved natural organic matter onto different aquifer materials: the effects of hydrophobicity and functional groups. Journal of Colloid and Interface Science 274, 380–391.

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