Michael H.B. Hayes1, Andre J. Simpson2, Guixue Song1, C. Edward Clapp3, and J. Burdon4

           1Chemicaland Environmental Sciences, Univerrsity of Limerick, Ireland;
           2Departmen of Physical and Environmental Sciences, University of Toronto at Scarborough, Canada;
           3USDA ARS, Department of Soil, Water and Climate, University of Minnesot, St. Paul, USA;
           4Department of Chemistry, University of Birmingham, England


   We follow the ‘Splitters’ approach for studies of Soil Humic Substances. In that we isolate and
 fractionate the components of Soil Organic Matter (SOM), as opposed to the ‘lumpers’ approach in
 which studies are carried out on the whole of the SOM.
   The classical methods for studies of SOM involves dissolving the OM in 0.1 or 0.5 M NaOH,                                                                                                                                                                                        FIG 6. Heteronuclear Multiple Quantum
 precipitating the humic acids at pH 1, recovering these and the soluble (in acid) fulvic acids. We                                                                                                                                                                                 Coherence Spectra (HMQC) of the DMSO
 introduce here novel procedures, based on charge density and polarity differences for the isolation and                                                                                                                                                                            FA UC. A) depicts the complete spectrum.
fractionation of the components of SOM, and characterization is carried out by solid and liquid state                                                                                                                                                                              Assignments in Figure 6A are as follows:
                                                                                                                                                                                                                                                                                   1, protons in p-hydroxybenzoates (lignin);
                                                                                                                                                                                                                                                                                   2, phenylalanine (in protein); 3, mainly
                                                                                                                                                                                                                                                                                    protons adjacent to an Ar-OR functionality
                                                                                                                                                                                                                                                                                   in lignin; 4, units in syringyl units (lignin);
 Experimental Procedures                                                                                                                                                                                                                                                           5, anomeric protons in carbohydrates;
                                                                                                                                                                                                                                                                                   6, other CH groups in carbohydrates;
                                                                                                                                                                                                                                                                                   7, CH2 in carbohydrates; 8, -protons in
                                                                                                                                                                                                                                                                                   proteins and peptides; 9, methoxyl in lignin;
   Soils were sequentially exhaustively extracted at pH 7, then 10.6, then 12.6, then with 0.1 M NaoH                                                                                                                                                                              10, aliphatic linkages including numerous
 + 6 M urea, then with DMSO + 6% concd. H2SO4, and the residual material was recovered after                                                                                                                                                                                       signals from various lipids, and side chain
destruction of the clays with HCl/HF. The aqueous extracts were processed by the XAD-8 and XAD-4                                                                                                                                                                                   protons in proteins/peptides.
resin in tandem procedure (Hayes, 2006). The DMSO was removed and the products recovered using
XAD-8 resin (Hayes, 2006). The samples were characterized and compared using solid and liquid state
                                                                                                                                                                                                                            B) shows an expansion of the aliphatic region.
                                                                                                                                                                                                                            Assignments in Figure B are as follows;
                                                                                                                                                                                                                            1, R-OCO-CH2-R methylene unit adjacent to
Results and Discussion                                                                                                                                                                                                      the carbonyl in lipids (including lipoproteins
                                                                                                                                                                                                                            and cutins); 2, methylene units in an aliphatic
                                                                                                                                                                                                                            chains  to an acid or ester; 3, methylene
                                                                                                                                                                                                                            (CH2)n in aliphatic chains, 4, methylene units
                                                                                                                                                                                                                            in an aliphatic chains  to an acid or ester;
                                                                                                                                                                                                                            5, CH3 (a small contribution in this region
                                                                                                                                                                                                                            will be from terminal CH3 from lipids).
                                                                                                                                                                                                                             However, the majority of signals are from
                                                                                                                                                                                                                            proteins. In proteins a classic elongated CH3
                                                                                                                                                                                                                            results as many of the side chain experience
                                                                                                             FIG 4 1H NMR spectra for A), the UREA FA Cultivated soil (C); B), the diffusion edited 1H                      varying 13C chemical shifts due to local
                                                                                                             spectrum of the UREA FA C; C), Bovine Serum Albumin; D), the diffusion edited 1H spectrum of the               environments with the macromolecular
                                                                                                             UREA HA C.                                                                                                     structure.

                                                                                                                “Simple” assignments, shown in A, indicate strong contributions from P, proteins/peptides; L,
                                                                                                             lignin; C, carbohydrate; WCL, waxes cuticles and lipids; PG, peptidoglycan; LP, lipoprotein. More
                                                                                                             specific assignments shown in B refer to 1, amide; 2, phenylalanine; 3, aromatics in lignin; 4,
                                                                                                             anomeric protons in carbohydrates; 5, -protons in proteins and peptides; 6, methoxyl in lignin; 7,
                                                                                                             other carbohydrate protons; 8, P-OCO-CH2-R methylene unit adjacent to the carbonyl in
                                                                                                             lipoprotein; 9, acetate group in peptidoglycan; 10, methylene units in an aliphatic chain  to an acid    Summary
FIG 1. CPMAS 13C NMR spectra of humic acids isolated at pH 7(1), 10.6 (2), 12.6 (3), and that                or ester; 11, methylene (CH2 )n in aliphatic chains; 12, CH3 (†note when this peak is large relative to
precipitated from dilute solution at pH 2 (4). Spectra 5, 6, and 7 are for the fulvic acids, and 8, 9,       11 it often indicates strong contributions from proteins as in these examples; see Figure 4C). In
                                                                                                             Figure 4C only the dominant protein signals are labeled. These when combined are the most
and 10 are for the XAD-4 acids isolated in the same pH sequence. Spectrum 11 is for neutrals                 distinctive indicators of protein in humic materials. In figure 4D, assignments are as above. Si
isolated in ethanol during soxhlet extraction of XAD-4.                                                      indicates a natural silicate species and not TMS (a commonly used NMR reference standard).                   FIG 1 shows that isolation of soil humic substances on the basis of charge density differences gives
                                                                                                                                                                                                                        rise to fractions of different compositions. In FIG 2 we see that the material removed in the urea is
   Note the definite compositional differences between the same fractions isolated at the different
                                                                                                                                                                                                                        similar to that isolated at pH 12.6 (after prior exhaustive extractions at pH 7 and 10.6). That
pH values. This shows the effectiveness of using charge density properties for the separation of                                                                                                                        indicates that materials held by ion exchange or by steric constraints are released in the urea
components of SOM.                                                                                                                                                                                                      solution. FIG 3 that exhaustive extractions in base + 6M urea effectively removes the humic
                                                                                                                                                                                                                        components derived from lignin. However, there is clearcut evidence for significant contributions
                                                                                                                                                                                                                        from aliphatic components (which are likely to contain long chain hydrocarbons, fatty acids, waxes,
                                                                                                                                                                                                                        etc.) and for carbohydrate and peptide functionalities in association with the soil clays.
                                                                                                                                                                                                                           When the humin components were further isolated in DMSO + 6% H2SO4, and fractionated into
                                                                                                                                                                                                                        materials that fall into the classical definitions of humic acids (but are not, of course such) we see in
                                                                                                                                                                                                                        the spectra in FIGS 4, 5, and 6 that the humin samples studied contain varying contributions from
                                                                                                                                                                                                                        five main categories of structures, namely protein, aliphatic species (including contributions from
                                                                                                                                                                                                                        lipoproteins), carbohydrates, peptidoglycan (the main structural components in bacterial cell walls)
                                                                                                                                                                                                                        and still some lignin-derived materials. Lignin is clearly identifiable as a plant input in humin
                                                                                                                                                                                                                        materials while peptidoglycan is indicative of microbial inputs. It is not possible in the present
                                                                                                                                                                                                                        studies to positively identify the source of the protein, aliphatic or carbohydrate species. In the case
                                                                                                                                                                                                                        of the lipids, a fraction would appear to be present as lipoproteins (which may be from lysed
                                                                                                                                                                                                                        microbial cells). Similarly, some of the carbohydrates can certainly be ascribed to microbial sources
                                                                                                                                                                                                                        (attributable to the carbohydrate backbone in peptidoglycan); however, from the size of N-acetyl
                                                                                                                                                                                                                        peak in peptidoglycan it is clear this peptidoglycan alone cannot account for all the carbohydrates

FIG 2. CPMAS 13C NMR spectra of humic acids isolated at pH 12.6 (0.1 M NaOH) after exhaustive
extractions at the lower pH values[lower], and for the HAs isolated in 0.1 M NaOH + 6 M urea 9                                                                                                                             The source of the other carbohydrates cannot be determined in this study and could include
(upper). Note the very close similarities in the spectra.                                                                                                                                                               structural carbohydrate from plants (cellulose, hemicellulose etc.) as well as other microbial
                                                                                                                                                                                                                        carbohydrates. The solubilised humin materials have components similar to those in the more
  These data suggest that the material isolated in the urea medium was held by hydrogen bonding                                                                                                                         traditional humic and fulvic acid fractions, with the exception that peptidoglycan is present at
and/or by entrapment within the humin matrix, and released by the urea.                                                                                                                                                 significant levels and these humin fractions have a macromolecular instead of supramolecular
                                                                                                                                                                                                                        character, such as for traditional HAs an FAs. That indicates that the humin fraction contains, in
                                                                                                                                                                                                                        addition to plant derived biomass, a significant amount of microbial biomass. Because the humin
                                                                                                                                                                                                                        fraction is strongly associated with clays, and because bacteria are known to associate strongly with
                                                                                                                                                                                                                        clay minerals, it is feasible that the clay component provides protection for microbial species that
                                                                                                                                                                                                                        ultimately contribute, in terms of biomass, to the operationally defined humin fraction. In addition,
                                                                                                                                                                                                                        it is feasible that some of the more labile components, especially proteins may be preserved through
                                                        FIG 3 CPMAS 13C NMR spectra of the clay                                                                                                                         associations with the clays. However further work will be needed to determine if that is the case, or
                                                        organic complex after exhaustive extraction                                                                                                                     if the protein is present due to the release of cellular proteins during lyses from DMSO and UREA.
                                                        with 0.1 M NaOH + 6 M urea. The spectrum is
                                                        contributed by classically defined humin

                                                                                                            FIG 5. 1H diffusion edited NMR of the A) UREA HA, uncultivated soil [UC]; B) DMSO HA UC; C)
                                                                                                            DMSO FA UC. Unless otherwise depicted, assignments are the same as shown in FIG 4. †Note this peak         Reference
                                                                                                            is too large to be attributed to aliphatic species alone, and indicates a substantial contribution from
                                                                                                               *It is very important to note the Humin HA is soluble in DMSO only in the presence of a strong acid.
   Note the highly significant aliphatic functionality (10-50 ppm), the –Oalkyl functionality (60-90 ppm;   D2SO4 was added which, in addition to solubilizing the humin, also deuterium exchanged the N-H (to N-
would include carbohydrate/peptide), the evidence for anomeric C (105 ppm), the evidence for the carbonyl                                                                                                              M.H.B. Hayes. 2006. Solvent systems for the isolation of organic components from soils. Soil Sci.
                                                                                                            D) and so the amide resonance in the humin HA is strongly attenuated.
of carboxyl (180 ppm), and the lack of significant aromatic functionality.                                                                                                                                             Soc. Am. J. 70, 986-994

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