Sequential exhaustive extraction of a Mollisol soil, and. characterizations of humic components, including humin, by

Transcription

1 Sequential exhaustive extraction of a Mollisol soil, and characterizations of humic components, including humin, by solid and solution state NMR G. SONG a, E. H. NOVOTNY ab, A. J. SIMPSON c, C. E. CLAPP d, M. H. B. HAYES a Short title: Characterization of humic/humin components by NMR a Department of Chemical & Environmental Sciences, University of Limerick, Limerick, Ireland, b Embrapa Solos, R. Jardim Botânico, 1024, CEP , Rio de Janeiro-RJ-Brazil, c Department of Physical and Environmental Sciences, University of Toronto, Scarborough Campus, Toronto, Ontario, Canada, M1C 1A4, and d Department of Soil, Water, and Climate, University of Minnesota, St Paul, MN 55108, USA Correspondence: M. H. B. Hayes. michael.h.hayes@ul.ie Received 1 March 2007; revised version received XXX, 2007 and accepted XXX,

2 Summary A comprehensive sequential extraction procedure was applied to isolate soil organic component using aqueous solvents at different ph values, base plus urea (base-urea), and finally dimethylsulfoxide (DMSO) plus concentrated H 2 SO 4 (DMSO-acid) for the humin-enriched clay separates. The extracts from base-urea, and DMSO-acid would be regarded as humin in the classical definitions. The fractions isolated from aqueous base, base-urea, and DMSO-acid were characterized by solid and solution state NMR spectroscopy. The base-urea solvent system isolated ca. 10% (by mass) additional humic substances. The combined base-urea and DMSO-acid solvents isolated ca. 93% of total organic carbon from the humin-enriched fine clay fraction (< 2 µm). Characterization of the humic fractions by solid-state NMR spectroscopy showed that oxidized char materials were concentrated in humic acids isolated at ph 7, and in the base-urea extract. Lignin-derived materials were in considerable abundance in the humic acids isolated at ph Only very small amounts of char-derived structures were contained in the fulvic acids and fulvic acids-like material isolated from the base-urea solvent. After extraction with base-urea, the 0.5 M NaOH extract from the humin-enriched clay was predominantly composed of aliphatic hydrocarbon groups, and with lesser amounts of aromatic carbon (probably including some char material), and carbohydrates and peptides. From the combination of solid and solution-state NMR spectroscopy, it is clear that the major components of humin materials, from the DMSO-acid solvent, after the exhaustive extraction sequence, were composed of microbial and plant derived components, mainly long-chain aliphatic species (including fatty acids/ester, waxes, lipids and cuticular material), carbohydrate, peptides/proteins, lignin derivatives, lipoprotein and peptidoglycan (major structural components in bacteria cell walls). Black carbon or char materials 2

3 were enriched in humic acids isolated at ph 7 and humic acids-like material isolated in the base-urea medium, indicating that urea can liberate char-derived material hydrogen bonded or trapped within the humin matrix. Introduction Soil organic matter, with its slow turnover of centuries to millennia (Balesdent & Mariotti, 1996) provides a relatively stable carbon pool in the global carbon cycle. Humic substances have a key role in the stable pool of soil organic matter since any depletion in their abundance will generate a large flow of CO 2 from the soil to the atmosphere. Humin is regarded as the most recalcitrant of the soil organic fractions and it persists longest in the soil environment. In the classical definitions humin is the fraction of humic substances insoluble in aqueous solutions at any ph value. Typically humin constitutes about half of the humic substances (HS) in soil (Stevenson, 1982), and more than 70% (by mass) of total organic matter in unlithified sediments (Hedges & Keil, 1995), and are considered to have significant resistance to transformations by microorganisms. Because it does not dissolve in the classical aqueous solvent systems used for the isolation of soil organic matter components, less is known about humin than about the other humified (or biologically or chemically transformed) or humic fractions that exist in soil, sediment or peat (Rice & MacCarthy, 1992). The chemical composition of this most recalcitrant fraction of soil organic matter is still a matter of debate. It has been variously described as high molecular weight, polymeric, a humic acid-clay complex, a humic acid intimately associated with inorganic soil colloids, a lignoprotein, a melanin or as plant and fungal 3

4 residues in varying stages of decomposition (Rice & MacCarthy, 1992, and references therein). A model by Rice & MacCarthy, based on extraction and fractionation using a methylisobutylketone (MIBK) solvent system (Rice & MacCarthy, 1989; 1991; 1992) suggests that the humin component consists of four fractions: a lipid fraction (referred to as bitumen ); a bound-lipid fraction; a bound humic acid ; and an insoluble residue usually with a large ash content. Recently, with the aid of modern analytical technologies, new models have been presented for humin fractions, such as: (1) an organo-mineral composite (Malekani et al., 1997); (2) selectively preserved, straight-chain, microbial biopolymers and apolar substances physically encapsulated by weak forces and by covalent bonds (Lichtfouse, 1999); (3) a partial supramolecular composition of trapped aliphatic hydrocarbons, proteins, covalently-bound fatty acids, and trapped fatty acids and/or esters as variously postulated also for humic acids (Burdon, 2001; Piccolo, 2001; Simpson, 2002; Guignard et al., 2005). The entrapments and associations would present difficulties for the isolation of the components in the associations or mixtures by means of the aqueous solvents on which the classical definitions for the humic components are based. Mild solvents are normally used to avoid chemical alteration of humic components. Dilute sodium hydroxide (0.1 M NaOH), as recommended for the isolation of the Standards of the International Humic Substances Society (IHSS), is used to isolate the humic acids (precipitated at ph 1 from the alkaline extracts of soil organic matter) and fulvic acids (soluble in water at all ph values) components (Swift, 1996). Then, to recover humin, HF/HCl is used to free the humin materials from the 4

5 inorganic soil colloids (Skjemstad et al., 1994; Preston & Newman, 1995; Schmidt et al., 1997; Wang & Xing, 2005). HF will remove most of the inorganic minerals, as well as paramagnetic species (such as Fe) allowing the humin fraction to be analysed by spectroscopic techniques. Some consider that the HF-HCl mixture can lead to hydrolysis and losses of polysaccharide and protein materials (Stevenson, 1982; Wang & Xing, 2005). The MIBK (methylisobutylketone) method, introduced by Rice & MacCarthy (1989) involves the partitioning of humin between an aqueous phase of varying ph and the MIBK layer. Almendros et al. (1996) used ultrasonic disaggregation followed by flotation in a bromoform-ethanol mixture and then partitioning in water-mibk to study soil humin. Alkyl components (56-81% of the total chromatographic area), including variable amounts of alkanes and fatty acids, are major constituents of the humin materials isolated using MIBK. Hayes (1985) listed criteria for good solvents for humic substances (see also Clapp et al., 2005) and considered that the best organic solvents for humic components have an electrostatic factor (EF) > 140 and pk HB values (indicating abilities to break hydrogen bonds) > 2. Acetone, though a powerful dipolar aprotic solvent for many organic chemicals, has EF (59.62) and pk HB (1.18) values outside these ranges and is not a good solvent for humic substances. Also its values for hydrogen bonding (δh) and proton accepter parameters (δb) are smaller than those considered to be good solvents for humic substances (Hayes, 1985; Clapp et al., 2005). However, Spaccini et al. (2000) and Spaccini et al. (2006) have extracted from soils a hydrophobic humic fraction by means of an acetone-hcl (8:2 by volume) solution, and these were shown by CP/MAS 13 C NMR spectroscopy to be greatly enriched in alky hydrocarbons. 5

6 Dimethylsulfoxide (DMSO), a dipolar aprotic solvent, is an excellent solvent for cations but a poor solvent for anions (Martin & Hauthal, 1975). It is a good hydrogen bond breaker and the non-polar (as distinct from the S=O face) stretch of DMSO can be considered to have affinities for less polar humin components. DMSO containing HCl (DMSO/HCl, 94:6 v/v) solvent has been studied for the isolation of humin-type material from a Mollisol (Clapp & Hayes, 1996), but Tsutsuki and Kuwatsuka (1992) and Zhu et al., (2005) found that an HCl acidified DMSO solvent system isolated less than 22% of total humin materials from different soils. In contrast, the combination of DMSO plus sulphuric acid (concentrated) is more effective than the DMSO/HCl solvent systems. Recent work in the laboratory of one of the authors (Simpson) has compared the amounts of total organic matter isolated with DMSO-d 6 (deuterated dimethylsulfoxide), containing small amounts of acids, such as trifluoroacetic acid (TFA) and D 2 SO 4, and characterized the extracts by means of solution-state proton NMR. When the solvent system contained 5% D 2 SO 4 (v/v), about 65% of the total organic carbon (TOC) was dissolved. This was almost three times that extracted in the solvent containing 10% TFA (v/v), and more than 30 times the amount extracted using traditional alkaline systems. Thus DMSO and H 2 SO 4 mixtures could provide a powerful solvent for humin fractions. Hayes (1985; 2006) reviewed the procedures and the principles involved in the isolation of HS from soils. Aqueous alkali solutions have been the traditional solvent systems since introduced by Achard (1786). Dissolution in aqueous base takes place when the conjugated bases of the acidic functionalities are solvated. Dissolution is impeded by the presence of divalent and polyvalent metals that form cationic bridges between the charged functionalities. Dissolution is inhibited in H + -exchanged systems as the result of inter- and intramolecular hydrogen bonding. Urea, a proton acceptor, 6

7 can be used as a hydrogen bond breaker for the extraction of humic substances, and solubilisation of H + -exchanged humic acids will take place in 5 M urea solution (Clapp et al., 2005). In this paper, we introduce novel procedures for the isolation of humin components from a Mollisol soil. A Mollisol was chosen because of its dark brown to black organic-rich surface horizon, and because these are rich in plant nutrients and are among the most fertile of the world s soils. Since a Mollisol provides the soil humic and fulvic acid standards of the IHSS, there is a considerable awareness of the compositions of the humic and fulvic acid components (see also Singer & Huang, 1991; Clapp & Hayes, 1999), but there is less awareness of the compositions of humin, the most recalcitrant of the humic fractions in these soils. For this study we have employed solid-state Variable Amplitude Cross- Polarization with Magic Angle Spinning (VACP/MAS) and Dipolar Dephasing (DD) 13 C NMR spectroscopy and solution-state one dimensional (1-D) and twodimensional (2-D) proton NMR spectroscopy to study the compositions of the humic substances. We adhere to the classical definitions for humin. However, the novel solvent sequence that we have used has isolated from such humin materials fractions that normally would fall within the definitions of humic and fulvic acids. Materials and methods Soil and soil treatments An Elliott silt loam Mollisol soil (Elliott silt loam, TOC 2.9 wt %) provided by the International Humic Substances Society (IHSS) was chosen for the study. The soil description and sampling site can be found elsewhere (IHSS website). The soil was H + -exchanged (1 M HCl), then washed with distilled water till chloride free, and the ph of the H + -exchanged soil was in the range

8 Extractions with aqueous base and base/urea solutions The soil was repeatedly extracted with 0.1 M NaOH at ph 7 until the optical density at 400 nm of the extract was less than 0.1. Then the process was repeated at ph 10.6, and finally with 0.1 M NaOH (ph 12.6). Details of the procedure were described by Hayes and Graham (2000). Exhaustive extraction was then carried out in the same way using 0.1 M NaOH + 6 M urea (base/urea). An atmosphere of N 2 gas was used for extractions at ph 10.6 and above. Extracts were centrifuged ( g, 30 minutes), the supernatant was adjusted to ph 7-8 (1 M HCl) and filtered twice through a 0.2-µm cellulose acetate membrane by means of compressed air at the pressure of ca. 137 kpa. The fine organic matter-rich clay from the pressure filter pads was collected, and added back for the next base/urea extraction. Extraction with 0.5 M NaOH Following the exhaustive extraction with the base/urea solvent system, the residual fine clay was dialysed against distilled water until the conductivity was less than 10 µs cm -1, and then freeze-dried. The clay was extracted with 0.5 M NaOH (under N 2 ) using a slight modification (i.e. the HCl/HF treatment was not used) of the IHSS procedure (Swift, 1996). The XAD-8 and XAD-4 resin-in-tandem procedure A modification of the XAD-8 [(poly)methylmethacrylate] and XAD-4 (styrenedivinylbenzene) resin in tandem technique (Malcolm & MacCarthy, 1992; Hayes & Graham, 2000) was used to isolate humic acid-like (HALM) and fulvic acidlike (FALM) materials from the base/urea extracts. The filtrate from the base/urea extracts was diluted with distilled water to a weak colour (an organic matter content < 15 mg Litre -1 ) and the ph was carefully 8

9 adjusted to 2.5 using 1 M HCl. The solutions were pumped slowly through the XAD-8 and XAD-4 resin columns in tandem (ca 30 to 40 ml minute -1 ). The resin columns were extensively washed with 0.01 M HCl, then with water. The XAD-8 column was then back eluted with 0.1 M NaOH, the centre cut eluate was adjusted to ph 1.5 (6 M HCl) and stored for 16 hours (or longer) at 4 C. The precipitated HALM was separated by siphoning, and by centrifuging ( g, 10 minutes), then re-dissolved in 0.1 M NaOH, and again diluted to a weak colour as before. This solution was again carefully adjusted to ph 2.5, pumped on to XAD-8, and the procedure outlined above was followed. Distilled water was passed through the column till the conductivity reached 100 µs cm -1, then the column was back eluted with 0.1 M NaOH and the eluate was passed through IR-120 (H + -exchanged) resin, then freeze-dried to give the humic acid-like material (HALM). The supernatant after precipitation of the centrecut material was filtered through a 0.2-µm membrane, then pumped on to XAD-8, washed with water till the conductivity reached 100 µs cm -1, back eluted in 0.1 M NaOH, passed through IR-120 resin and freeze-dried to give the fulvic acid-like material (FALM). Materials retained on the XAD-4 resin were recovered in the same way as the FALM to give the XAD-4 acids. These acids were not used in the work reported here. Extraction and fractionation of DMSO humin fractions Fine clays were recovered on the pressure filtration pads as mentioned above during the recovery of the base/urea and the 0.5 M NaOH extracts. The clay was dialyzed to remove the base and urea, and then freeze-dried. Dried humin-rich clays in dry 500 ml glass containers were stirred by using magnetic followers for 14 hours in DMSO + 6% H 2 SO 4 (98 wt%) (DMSO/acid, 94:6 by volume; clay/solvent ratio 1:10). Clay and DMSO mixtures were centrifuged (15 9

10 000 g, 30 minutes). The extraction was repeated until the colour of the extract was negligible. Water was added to the DMSO/acid extract until the ph reached 2. The solution was stored at 4 o C to allow precipitation to take place. The precipitate (DMSO humin) was recovered after centrifuging ( g, 10 minutes), and washed four times with distilled water, then dialysed against distilled water, and freeze-dried. Determination of TOC of clay samples The Walkey-Black dichromate oxidation method (Allison, 1965) was used to determine the TOC of the clay samples at the various stages of extractions. VACP/MAS NMR 13 C spectroscopy Solid-state 13 C nuclear magnetic resonances (NMR) experiments were carried out by means of a Varian Inova spectrometer (Varian, Palo Alto, California, USA) at 13 C and 1 H frequencies of and MHz, respectively. Jackobsen 5 mm MAS doubleresonance probe heads were employed. The Variable Amplitude Cross-Polarization with Magic Angle Spinning technique (VACP/MAS) was applied with a contact time of 1 ms, a spinning speed of 13 khz, acquisition times of 13 ms, recycle delays of 500 ms. Spectra were divided into chemical shift regions as follows: 0-46 ppm, alkyl C; ppm, methoxyl and N-alkyl C; ppm, O-alkyl C; ppm, di-o-alkyl (anomeric) C and some aromatics; ppm, aromatic C; ppm, O- aromatic C; ppm, carboxyl, amide and ester C, and ppm, carbonyl C. Areas of the chemical shift regions were determined after integration and expressed as percentages of total area ("relative intensity"). The Dipolar Dephasing (DD) experiments were carried out with a dipolar dephasing time of 67 µs in order to distinguish between protonated C in rigid structures and non-protonated or mobile carbons. Due to coupling between 13 C and neighboring 1 H, the signals of C species 10

11 with strong dipolar interactions will vanish after the dipolar dephasing time, while those of C species with weak dipolar interactions will still be visible in the spectrum (Opella & Frey, 1979; Knicker et al., 2005b). The areas relative to these resonance intervals were used to evaluate the percentage of Aromaticity and Aliphaticity as follows: Aromaticity = [Aromatic C peak ( ppm)] 100/[Total peak area (0-230 ppm)]; Aliphaticity = 100- Aromaticity. 1-D 1 H NMR and diffusion edited spectroscopy NMR data were obtained by means of a Bruker Avance 500 MHz spectrometer (Bruker, Karlsruhe, Germany) employing a 1 H-BB- 13 C TBI probe fitted with an actively shielded Z gradient. One dimensional (1-D) solution state 1 H NMR experiments were carried out with 512 scans, a recycle delay of 3 s, and 32 K time domain points. Spectra were apodized through multiplication with an exponential decay corresponding to 1 Hz line broadening in the transformed spectrum, and a zero filling factor of 2. Diffusion Edited Experiments were carried out with a bipolar pulse longitudinal encode-decode sequence (Wu et al., 1995; Simpson et al., 2003b). Scans (1024) were collected using a 1.25 ms, 53.5 Gauss/cm, sine shaped gradient pulse, a diffusion time of 50 ms, 8192 time domain points and a sample temperature of 298 K. Spectra were apodized through multiplication with an exponential decay corresponding to 1 Hz line broadening in the transformed spectrum, and a zero filling factor of 2. 11

12 2-D 1 H- 13 C TOCSY and HMQC spectroscopy TOtal Correlation SpectroscopY (TOCSY) spectra were acquired in the phase sensitive mode, using time proportional phase incrimination (TPPI). Scans (512) were collected for each of the 256 increments in the F1 dimension. 2K data points were collected in F2, a mixing time of 50 ms and relaxation delay of 2 s were employed. Both dimensions were processed using sine-squared functions with a π/2 phase shifts and a zero-filling factor of 2. Heteronuclear Multiple Quantum Coherence (HMQC) spectra were collected in phase sensitive mode using Echo/Antiecho gradient selection; 512 scans were collected for each of the 256 increments in the F1 dimension; 2 K data points were collected in F2, a 1 J 1 H- 13 C (145 Hz) and a relaxation delay of 2 s was employed. The F2 planes were multiplied by an exponential function corresponding to a 5 Hz line broadening, while the F1 dimension was processed using sine-squared functions with a π/2 phase shift and a zero-filling factor of 2. Results and discussion The 0.1 M NaOH + 6 M urea extracted an additional 10% (by mass) organic matter from the soil residue that culminated in the extraction at ph The fine clay, isolated after extraction with the base/urea solvent, had a TOC content of 7.1% (by mass). After the subsequent extractions (0.5 M NaOH, and DMSO + 6% v/v H 2 SO 4 ), the TOC content was 0.5%; thus ~93% of humin material was isolated from the clay by this solvent sequence. 12

13 Solid state NMR spectroscopy Humic acids, fulvic acids, HALM and FALM components The VACP/MAS 13 C NMR spectra of humic acids, fulvic acids, HALM and FALM from extracts at ph 7, 12.6, and in the base/urea system are shown in Figure 1. The spectra have signals for alkyl C (23, 29 ppm), O alkyl C (56, 72 ppm), di O alkyl ( ppm), carboxyl ( ppm), and aromatic C ( ppm). Some O substituted aromatic carbon ( ppm) could be derived from lignin units. In the aliphatic C region, the peaks at about 23 and 29 ppm are from terminal methyl groups and from methylene carbons in aliphatic rings or chains. In the oxygenated aliphatic C region, the peak at 56 ppm can be assigned to methoxyl associated with lignin and lignin-like material as revealed also in the corresponding DD spectra, and N alkyl (α- C of most amino acids) from protein/peptides. The signal at 56 ppm was greatly attenuated in the DD spectra because only mobile methoxyl C (and not protonated N alkyl in protein/peptides) survived. The peak at 72 ppm can be attributed to polysaccharide structures, and the peak at ppm indicates anomeric C (di O alkyl) in carbohydrates. A doublet O aryl signal near 150 ppm with the great aryl C peak near 130 ppm, with a small signal near 115 ppm, as well as the resonances at 56 ppm, are characteristic of lignin-derived units, indicating the aryl C contribution in the sample is largely from lignin or lignin-like materials. The large aryl C signal at 129 ppm, which persists in the DD spectra, and with no distinct peaks near 150 or 120 ppm, is typically assigned to black carbon (BC) (hydrogen-deficient condensed aromatic structures) or charred material (Skjemstad et al., 2002; Novotny et al., 2006). The spectrum of humic acids isolated from ph 7 (Figure 1a) show prominent resonances in the ppm range, and there are clear, though significantly lesser resonances from aliphatic methylene, terminal methyl carbons, and O alkyl C 13

14 (including carbohydrate). A significant part (~75%) of the aromatic carbons is unprotonated, since the signal persists in the DD spectrum (Figure 1a). The humic components derived from black carbon are characterized by hydrogen-deficient condensed aromatic structures and great charge densities attributable to carboxylic groups linked to the aromatic core (Kramer et al., 2004; Novotny et al., 2007). The aromatic carbons of BC or char are dominated by the 110 to 140 ppm resonance (Clapp & Hayes, 1999; Schmidt et al., 1999; Skjemstad et al., 2002; Song et al., 2002; Simpson & Hatcher, 2004; Knicker et al., 2005a; Novotny et al., 2006). The carboxylated condensed aryl carbons (VACP and DD in Figure 1a) are consistent with the acidic properties of this fraction (ph 7 extract). To be soluble at ph 7, the condensed aryl compounds must contain a significant charge density. This arose from the partial oxidation of BC resulting in a stable (condensed aryl) and reactive (rich in COO - ) functionalities. Additionally, fulvic acids isolated at ph 7 had greater charge densities (more carboxyl) and more hydrophilic polar substituents, including carbohydrates, than the humic acids at ph 7 (Figures 1a and 1d, and Table 1). However, the DD spectrum (Figure 1d and Table 1) shows more hydrogen substituted aromatic carbons and hence the major char components were concentrated in the humic acids fraction. There are significant differences in the NMR spectra for the humic and fulvic acid fractions from the extracts of ph 7 and at ph 12.6 (Figures 1a-1b and 1d-1e). The major differences between the VACP spectra for the extracts at ph 7 and ph 12.6 are in the aromatic ( ppm), and O substituted aromatic ( ppm) resonances. These resonances for the ph 7 extracts are characteristic of Mollisol humic and fulvic fractions, and more especially those of the humic acids. The aryl profile is typical of charcoal or char-derived materials (as evidenced by the great 14

15 recovery of aryl C following chemical oxidation) (Skjemstad et al., 2002). Aromatic carbons composed 48% (Table 1) of the humic acids spectrum of the isolate at ph 7 (Figure 1a), and aromaticity decreased to 33% for the extract at ph Prominent peaks in the humic acids spectrum at 56, 130, and 151, ppm, and the doublet at around 151 ppm (Figure 1b) indicate definite contributions from lignin or lignin-derived materials (Hatcher, 1987). Aromaticities of the fulvic acids were much less than for the corresponding humic acids, and the DD spectra indicate that most of the aromatic components had more hydrogen substituents (Figures 1d and 1e), and especially in the case of the isolate at ph 12.6 (Figure 1e). Aliphaticity increased with increasing extractant ph (Table 1). The fulvic acids isolated at ph 7 were more oxygenated than those in the humic acids, as indicated by the relatively greater abundances of the carboxylic and carbohydrate carbons than for the humic acids isolated at the same ph. The evidence for origins in lignin in the cases of fulvic acids and humic acids isolated at ph 12.6 is greatly enhanced by the strong resonances at 56 and at 146 ppm (Figures 1b and1e) and the fulvic acid fraction was especially enriched in carbohydrates (resonances at 72 and at 103 ppm). The DD spectrum would suggest that char did not make a significant contribution to fulvic acids isolated at ph There are important similarities between the VACP spectrum for the HALM isolated in base/urea (Figure 1c) and the humic acids isolated at ph 7 (Figure 1a). In the classical definitions these extracts would be regarded as humin materials. However, the similarities of VACP spectra in Figures 1a and 1c suggest that the inclusion of urea released char containing humic acids held by hydrogen bonding, and/or perhaps by steric constraints within the humin matrix. A study by Skjemstad et al. (2002) indicated that the charcoal content in the Elliott silt loam Mollisol was ~ 6.6 g C kg -1 or 23% of the soil TOC by mass. Using a procedure that combined acid 15

16 demineralisation, base extraction, and dichromate oxidation, Song et al. (2002) isolated black carbon and kerogen in amounts ranging from 18.3% to 41.0% of the TOC in soils and sediments from an industrial region in China. They considered that fine carbon particles may have been degraded and weathered chemically and biologically and were closely associated with clay surfaces and/or encapsulated in the humin fraction. The VACP spectrum of the FALM isolated in the urea system has closer similarities to the fulvic acids isolated at ph 12.6 than those isolated at ph 7 (Figure 1f). Aromaticity was least in this fraction, and the DD spectrum suggests considerable hydrogen substitution in the aromatic structures (hence a negligible contribution from char materials). The carbohydrate content was large (resonances at 72 and 102 ppm), and the resonance at 56 ppm is more likely to arise from peptide than from methoxyl functionalities. Also, the DD spectrum (Figure 1f) indicated small contributions from lignin residues in this fraction. HALM from 0.5 M NaOH The VACP and DD spectra in Figure 2 indicate that long-chain aliphatic hydrocarbon functionalities were the major contributors to the compositions of the materials in 0.5 M NaOH from the fine clays that had been extracted in the base/urea system. Comparisons of the VACP and DD spectra, and the data in Table 1 would suggest that some additional char materials were released. Contributions from lignin were minor. The resonance at 30 ppm is typically assigned to mobile or amorphous polymethylene in long-chain aliphatic groups, such as resistant biopolymers (waxes, lipid including lipoprotein or cutins, suberans, fatty acids/esters). It is probable that much of the significant carboxyl/ester functionality was derived from aliphatic 16

17 acids/esters, and from oxidized char materials. The strong base increased the amounts extracted significantly, but its use cannot be recommended because of the dangers of oxidations of the phenolic (from lignin) structures in particular (Clapp et al., 2005, p32 and references therein). DMSO humin A mixture of DMSO + 6% H 2 SO 4 (v/v) was used to solubilize the most recalcitrant humin materials. The VACP/MAS NMR spectrum of the DMSO humin is shown in Figure 3. This sample was not treated with HF; thus the ash content was large (~15 %). The spectrum is dominated by strong resonances from the terminal methyl and from the polymethylene carbons of aliphatic species (0 46 ppm). The major peak at 30 ppm indicates long-chain aliphatic structures in the DMSO humin. Note the small peak of methoxyl or N alkyl from peptides at 56 ppm, the contribution from carbohydrate at 72 ppm (confirmed by the anomeric carbon at 105 ppm), the relatively small aromatic carbon content at ppm, and the sharp resonances from carboxylic, esters, amide carbons at 172 ppm. This DMSO humin fraction has features similar to other humin fractions described in the literature (Hatcher et al., 1980; Preston & Newman, 1995; Fabbri et al., 1998; Hu et al., 2000; Song et al., 2005; Wang & Xing, 2005). The amorphous polymethylene carbons could be assigned to the strong sharp peak at 30 ppm (Kögel-Knabner et al., 1992b; Hu et al., 2000). This alkyl carbon in humin could be derived from selectively preserved plant polyesters of cutin and other aliphatic biopolymers during the humification process. The peak at 33 ppm, that is normally assigned to crystalline polymethylene carbons, was not apparent in the case of the Mollisol DMSO humin (Figure 4). However, clay-humin associations can 17

18 preserve ordinarily labile compounds, such as carbohydrates and proteinaceous material. The spectrum for the DMSO humin extract from the Mollisol was similar to those for the humin residues in several Irish grassland soils after DMSO/acid extraction had been carried out (data not shown). We have not found evidence for significant amounts of BC or char in the Irish grassland soils. Thus it would appear that the majority of the char materials had been removed from the clay and associated organic materials by the sequential extractions of the Mollisol, culminating in the base/urea system. Some of this material could, of course, have remained in the residual humin, and that could be accessed after HF treatment. Brodowski (2006) found the greatest concentrations of BC in the < 53 µm size fraction and in the occluded particulate fractions, and it was considered that, in preference to other organic materials, BC was embedded in microaggregates. The base/urea solvent system would effectively breakdown the clay-humic associations and release char type components, as evidenced in Figure 1c. The properties of DMSO and of DMSO in association with H 2 SO 4 (Hayes 1985, 2006; Clapp et al., 2005) can explain the efficacy of that solvent system for dissolving the non-polar humin materials evidenced in Figure 3. With the exception of the HALM from the base/urea system, aromaticity of the humic and fulvic acids decreased and aliphaticity increased for the extracts obtained by the solvent sequence used (Table 1). Aromaticity was greatest where evidence was strongest for char-derived humic acids. The smallest percentage of aromatic carbons and the greatest aliphaticity were in DMSO humin. That emphasized the predominantly aliphatic compositions of humin components. 18

19 Solution-state NMR spectroscopy 1 H NMR spectroscopy The 1-D 1 H spectra had large water/urea signals in most cases and for that reason diffusion edited spectra are shown in Figure 4. The 1-D 1 H NMR spectra (not shown) of HALM and DMSO humin are similar to the diffusion edited 1 H NMR spectra, except that some signals of small molecules are greatly attenuated. The spectral regions in 1 H NMR have been assigned (Simpson et al., 2003a; Simpson et al., 2007). General regions (Figure 4b) highlighted by brackets are applicable to all 1 H NMR and diffusion edited 1 H NMR spectra, and can be defined broadly as: (1) mainly aromatic and amide; (2) signals from a numerous moieties including carbohydrate, peptides, lignins, etc.; (3) aliphatic signals from different chemical environments including various substituted methylenes; α, β protons to a functionality in hydrocarbons, lipoprotein (LP), peptidoglycan (PG); long-chain methylene in lipids, waxes, cuticles, etc.; terminal CH 3 groups. More specific assignments are presented in Figure 4a. Diffusion editing spatially encodes molecules at the start of the experiment and then refocuses these at the end of the experiment. Species that diffuse or exhibit a great degree of motion during the experiment are not refocused and are essentially gated from the final spectrum. In essence, the spectrum produced will contain only signals from species that undergo little or no self-diffusion; hence structures identified will be macromolecular or in rigid domains, or both. The majority of the humin signals remained after diffusion editing (Simpson et al., 2007). The diffusion gate employed in this study was carried out using 63 Gauss encoding and decoding gradients (the instrumental maximum). In other studies employing similar experimental conditions, nearly all signals are destroyed and only those from macromolecules survive (Kelleher et al., 2006). Similarly, in classic humic and fulvic 19

20 materials, the vast majority of signals would be greatly attenuated under these conditions. This disparity tells us that the components in the humin fraction are of much greater size than those previously seen in fulvic and humic acids extracts, and are likely to be composed of macromolecules and/or very stable rigid aggregates. HALM dissolved in DMSO-d 6 and, in the case of DMSO humin, 4% D 2 SO 4 (deuterated sulphuric acid) was added to the sample to ensure complete solubilisation. The HALM spectrum is dominated by contributions from peptides (see Figure 4a; the double hump at ~ ppm is indicative of peptides, especially when there is a large methyl resonance at ~ 0.8 ppm, and H-N resonances are also present at ~ ppm), from cutins, waxes, and/or lipids, and lignin/carbohydrate near ppm, all of which have previously been identified in classic humic and fulvic acid fractions (Simpson et al., 2002; Kelleher & Simpson, 2006). The spectrum of the DMSO humin (Figure 4b) is dominated by the presence of the large contribution from longchain (CH 2 ) n. The ratio observed between the main chain (CH 2 ) n and methylene units in an aliphatic chain, β to an acid or ester, is closer to that in aliphatic biopolymers, such as cutins, and similar to that observed for the other humic fractions (Simpson et al., 2003a). This is consistent with the presence of aliphatic chains observed by solidstate VACP/MAS NMR spectroscopy (Figure 3), which would be explained by the highly aliphatic nature of humin. Different sources have been considered for the origins and formation pathways of aliphatic moieties in the humin fraction or stable aggregates, such as largely aliphatic, resistant macromolecular components from cuticular material and suberins, or moieties with long alkyl chains derived from the cross-linking of lipids and/or cutin and suberin polyesters, and from soil microorganisms (Almendros & Sanz, 1992; Kögel-Knabner et al., 1992a; Lichtfouse et al., 1998; Poirier et al., 2000; Simpson et 20

21 al., 2007). The contribution of abundant aliphatic moieties to the recalcitrant fraction of humin is thus well recognised. There are also considerable aromatic signals present in both the solid (Figure 3) and solution state NMR spectra (Figure 4). These are likely to be from lignin components, and from aromatic amino acids that have been identified in the 2 D NMR of similar materials (Simpson et al., 2007). The contribution from char or BC to these samples cannot be ruled out because condensed aromatic structures would resonate in the region labelled 1 in Figure 4a (Simpson et al., 2007). It is difficult, using solution-state NMR spectroscopy only, to definitively assign the broad resonance from ~ ppm in Figure 4a to either residual amide signals or to other chemical structures that may include condensed aromatic rings. However, the aromatic signal in the VACP spectrum, which persists in the DD experiment (Figure 1c), indicates the presence of condensed aromatic structures in this fraction. 2-D NMR spectroscopy of DMSO humin Two-dimensional NMR spectroscopy offers significant advantages for the analyses of complex humic structures. An increased signal dispersion into two frequency dimensions greatly reduces resonance overlap. Cross peaks in 2-D NMR spectra indicate a range of connectivities defined by the kind of NMR experiment carried out, allowing a probe of bonding interactions, spatial relationships, and chemical exchanges (Simpson, 2001). Total Correlation Spectroscopy (TOCSY) is used to identify coupling between protons that are connected via a bonded network, and are very useful for the study of proton connectivity in humic substances. TOCSY peaks 21

22 arise from the interactions of protons over numerous bonds, according the experimental setup. Figure 5 shows the TOCSY spectra of the DMSO humin. The proton couplings from the major categories present have been highlighted. Major connectivities can be summarized as: coupling from amides in peptides; coupling from double bonds from aromatic structures; coupling between α protons and amino acids side chains in peptides/proteins; coupling between aliphatic compounds; and between aliphatic alcohols and ethers. Full interpretation of all of the cross peak can be made only by combining information from a range of NMR techniques. More detailed assignments can be found in the literature (Fan et al., 2000; Simpson, 2001; Kelleher & Simpson, 2006). Heteronuclear multiple quantum coherence (HMQC) experiments are applied widely to study single H-C bond correlations in humic substances. Heteronuclear 2-D spectra do not exhibit a spectrum diagonal. Cross-peaks arise from coupling of a 13 C with a 1 H rather than coupling between adjacent protons. A 1 H bonded directly to a 13 C atom will produce a cross-peak at the point of interaction of the 1 H and 13 C chemical shifts (Simpson, 2002). An expansion of the aliphatic and the central regions of the spectrum of HMQC in the ppm region of the Mollisol DMSO humin is shown in Figure 6. Many components of the DMSO humin fractions that are masked or overlapped by 1 H NMR and TOCSY are separated in the HMQC spectra. Region 1 in Figure 6 is crowded with aliphatic linkages, such as numerous signals from lipids and side chain protons in proteins/peptides. The resonances at around 10~25 ppm in F2 ( 13 C) and 0.5~0.9 ppm in F1 ( 1 H) result partly from terminal methyl groups from lipids. However, the majority of signals could be from proteins/peptides. The methylene (CH 2 ) n in aliphatic 22

23 chains and methylene units in aliphatic chains β and γ to acids or esters show strong contributions in the region of 0.9 ~ 2 ppm (F1) and 25~45 ppm (F2). Note the small hump at region 1 is assigned to the N acetyl group in peptidoglycan, the main component in bacterial cell walls. There is little peptidoglycan in the HALM of the Mollisol, but it is clear in the DMSO humin fraction (Figure 4b, Figure 6). Peptidoglycan was reported in the natural organic matter colloidal fractions from river waters (Croue, 2004; Wershaw et al., 2005). Label 2 in Figure 6 may represent components mainly from lipoprotein and signals from fatty acid/cuticule materials. Labels 3 and 4 are resonances from CH 2 and CH groups of carbohydrates. A small contribution results from methoxyl in lignin-derived structures, and the α protons in peptides/proteins. Peptidoglycan can make contributions to the resonances from methyl, carbohydrate and peptides functionalities. Resonances from the α-proton in peptides (in the region 4 to 4.5 ppm) show two distinct cross peaks in Figure 6. That is consistent with the data in the 1-D spectrum (Figure 4). Conclusions We have shown that: (1), A comprehensive sequential extraction procedure isolated humic acids, fulvic acids, and humin fractions from a Mollisol soil. (2), The highly oxidized char components were concentrated in humic acids isolated at ph 7, and lignin-derived components were concentrated in humic acids isolated at ph There was little evidence for char materials in fulvic acids isolated at the higher ph. The humic acids isolated at ph 12.6 had lignin-derived materials as major structural components, and there was significant lignin-derived material in the fulvic acids isolated at ph

24 (3), Contrary to expectation, the humic acids isolated in base/urea solvent were very similar to the humic acids isolated at ph 7. That indicates that urea librated charenriched humic acids that were hydrogen bonded or trapped within the humin matrix. Fulvic acids isolated in base/urea solvent were similar to fulvic acids isolated at ph (4), Additional humic acid-like materials released with 0.5 M NaOH from the fine clay fraction showed some lignin character. However, it was largely composed of long-chain aliphatic moieties, and with evidence for peptides and carbohydrates. (5), More detailed information from solution-state NMR spectra has clearly indicated compositional differences between humin materials isolated (after the previous exhaustive extractions) from base/urea and DMSO/acid extracts. A multiplicity of functional groups is evident in the solution-state NMR spectra, with clear evidence for lignin-derived structures, for carbohydrates, for peptides, for peptidoglycan and for aliphatic hydrocarbon structures. That indicates that the major components of the DMSO humin are carbohydrate, peptide, lignin-derived material, lipoprotein, fatty acids, cuticular material, peptidoglycan. The dominant species contained long-chain methylene and methyl groups. Theses functionalities were confirmed by 2-D TOCSY and HMQC spectra. (6), The data have shown that the humin materials are composed of microbial and plant derived components. (7), The techniques used clearly show that humin can be isolated from soil and give materials amenable to detailed compositional studies by means of solid and solution-state NMR spectroscopy. 24

25 Acknowledgements We acknowledge the support from Science Foundation Ireland (SFI), Environmental Protection Agency (EPA) Ireland, and the Irish Research Council for Science, Engineering and Technology (IRCSET). We thank Dr Eduardo R. deazevedo and Professor Tito J. Bonagamba for solid-state NMR data, and Dr Emma Simth, Buum Lam for assistance for acquisition of solution-state NMR data, and our colleagues, Raymond McInerney, Corinna Byrne for their assistance in the laboratory. References Aachard, F.K Chemische Utersuchung des Torfs. Crell's Chemische Annalen, 2, Allison, L.E Organic carbon. In: Method of Soil Analysis, Part 2, Chemical and Microbiological Properties (eds. Black, C.A., Evans, D.D., White, J.L., Ensminger, L.E. & Clark, F.E.), pp American Society of Agronomy, Inc., Madison, WI, USA. Almendros, G., Guadalix, M.E., Gonzalez-Vila, F.J. & Martin, F Preservation of aliphatic macromolecules in soil humins. Organic Geochemistry, 24, Almendros, G. & Sanz, J A structural study of alkyl polymers in soil after perborate degradation of humin. Geoderma, 53,

26 Balesdent, J. & Mariotti, A Measurement of soil organic matter turn over using 13-C natural abundance. In: Mass Spectrometry of Soils (eds. Boutton, T.W. & Yamasaki, S.I.), pp Marcel Dekker, New York. Brodowski, S., John, B., Flessa, H. & Amelung, W Aggregate-occluded black carbon in soil. European Journal of Soil Science, 57, Burdon, J Are the traditional concepts of the structures of humic substances realistic? Soil Science, 166, Clapp, C.E. & Hayes, M.H.B Isolation of Humic Substance from an Agriculture Soil Using a Sequential and Exhaustive Extraction Process. In: Humic Substances and Organic Matter in Soil and Water Environments: Characterization, Transformations and Interactions (eds. Clapp, C.E., Hayes, M.H.B., Senesi, N. & Griffith, S.M.), pp International Humic Substances Society, St Paul, Minnesota, USA. Clapp, C.E. & Hayes, M.H.B Characterization of humic substances isolated from clay- and silt-sized fractions of a corn residue-amended agricultural soil. Soil Science, 164, Clapp, C.E., Hayes, M.H.B., A.J.Simpson & Kingery, W.L Chemistry of Soil Organic Matter. In: Chemical Processes in Soils (eds. Tabatabai, M.A. & Slarks, D.L.), pp Special Publication, No.8. Soil Science Society of America, Madison, WI. Croue, J Isolation of humic and non-humic NOM fractions: structural characterization. Environmental Monitoring and Assessment, 92, Fabbri, D., Mongardi, M., Montanari, L., Galletti, G.C., Chiavari, G. & Scotti, R Comparison between CP/MAS 13 C-NMR and pyrolysis-gc/ms in the 26

27 structural characterization of humins and humic acids of soil and sediments. Journal of Analytical Chemistry, 362, Fan, T.W.M., Higashi, R.M. & Lane, A.N Chemical Characterization of a Chelator-Treated Soil Humate by Solution-State Multinuclear Two- Dimensional NMR with FTIR and Pyrolysis-GCMS. Environmental Science and Technology, 34, Guignard, C., Lemee, L. & Ambles, A Lipid constituents of peat humic acids and humin. Distinction from directly extractable bitumen components using TMAH and TEAAc thermochemolysis. Organic Geochemistry, 36, Hatcher, P.G Chemical structural studies of natural lignin by dipolar dephasing solid-state 13 C nuclear magnetic resonance. Organic Geochemistry, 11, Hatcher, P.G., VanderHart, D.L. & Earl, W.L Use of solid-state 13 C NMR in structural studies of humic acids and humin from Holocene sediments. Organic Geochemistry, 2, Hayes, M.H.B Extraction of Humic Substances from Soil In: Humic Substances in Soil, Sediment, and Water: Geochemistry, Isolation, and Characterization (eds. Aiken, G.R., McKnight, D.M., Wershaw, R.L. & MacCarthy, P.), pp John Wiley & Sons, New York. Hayes, M.H.B Solvent Systems for the Isolation of Organic Components from Soils Soil Science Society of America Journal, 70, Hayes, M.H.B. & Graham, C.L Procedures for the isolation and fractionation of humic substances In: Humic Substances. Versatile Components of Plants, Soils and Waters. (eds. Davies, G. & Ghabbour, E.A.), pp The Royal Society of Chemistry, Cambridge. 27

28 Hedges, J.I. & Keil, R.G Sedimentary organic-matter preservation: an assessment and speculative synthesis. Marine Chemistry, 49, Hu, W.G., Mao, J., Xing, B. & Schmidt-Rohr, K Poly(methylene) crystallites in humic substances detected by nuclear magnetic resonance. Environmental Science and Technology, 34, International Humic Substances Society IHSS Bulk Source Materials. available: [Accessed, 30 Jan 2007, 2007] Kelleher, B.P. & Simpson, A.J Humic substances in soils: are they really chemically distinct? Environmental Science and Technology, 40, Kelleher, B.P., Simpson, M.J. & Simpson, A.J Assessing the fate and transformation of plant residues in the terrestrial environment using HR-MAS NMR spectroscopy. Geochimica et Cosmochimica Acta, 70, Knicker, H., Gonzalez-Vila, F.J., Polvillo, O., Gonzalez, J.A. & Almendros, G. 2005a. Fire-induced transformation of C- and N-forms in different organic soil fractions from a Dystric Cambisol under a Mediterranean pine forest (Pinus pinaster). Soil Biology and Biochemistry, 37, Knicker, H., Totsche, K.U., Almendros, G. & Gonzlez-Vila, F.J. 2005b. Condensation degree of burnt peat and plant residues and the reliability of solid-state VACP MAS 13 C NMR spectra obtained from pyrogenic humic material. Organic Geochemistry, 36, Kögel-Knabner, I., de Leeuw, J.W. & Hatcher, P.G. 1992a. Nature and distribution of alkyl carbon in forest soil profiles: implications for the origin and humification of aliphatic biomacromolecules. Science of The Total Environment, 117/118,

29 Kögel-Knabner, I., Hatcher, P.G., Tegelaar, E.W. & de Leeuw, J.W. 1992b. Aliphatic components of forest soil organic matter as determined by solid-state 13 C NMR and analytical pyrolysis. Science of The Total Environment, 113, Kramer, R.W., Kujawinski, E.B. & Hatcher, P.G Identification of black carbon derived structures in a volcanic ash soil humic acid by Fourier transform ion cyclotron resonance mass spectrometry. Environmental Science and Technology, 38, Lichtfouse, E A novel model of humin : Humic substances. Analusis, 27, Lichtfouse, E., Chenu, C., Baudin, F., Leblond, C., Da Silva, M., Behar, F., Derenne, S., Largeau, C., Wehrung, P. & Albrecht, P A novel pathway of soil organic matter formation by selective preservation of resistant straight-chain biopolymers: chemical and isotope evidence. Organic Geochemistry, 28, Malcolm, R.L. & MacCarthy, P Quantitative evalutation of XAD-8 and XAD- 4 resins used in tandem for removing organic solutes from water. Environment International, 18, Malekani, K., Rice, J.A. & Lin, J.-S The effect of sequential removal of organic matter on the surface morphology of humin. Soil Science, 162, Martin, D. & Hauthal, H.G Dimethyl Sulphoxide. Van Nostrand Reinhold, New York. Novotny, E.H., deazevedo, E.R., Bonagamba, T.J., Cunha, T.J.F., Madari, B.E., Benites, V.d. & Hayes, M.H.B Studies of the compositions of humic 29