The Nature of Organic Materials in Intimate Associations with the Soil Clay Fraction

Transcription

1 The Nature of Organic Materials in Intimate Associations with the Soil Clay Fraction Michael H.B. Hayes 1, Andre J. Simpson 2, Guixue Song 1 1 Chemical and Environmental Sciences University of Limerick, Ireland 2 Department of Chemistry University of Toronto at Scarborough Toronto, Canada

2 Classical Definitions Humus: Components of Soil Organic Matter so transformed as to bear no morphological resemblances to the materials of origin Humic Substances: Amorphous, brown, polymeric substances that are differentiated on the basis of solubility properties into: HUMIC ACIDS, Precipitated at ph 1 from solution in aqueous base; FULVIC ACIDS, soluble in aqueous media at all ph values; HUMIN, insoluble in aqueous media

3 COMPOUNDS BELONGING TO RECOGNISABLE CLASSES, SUCH AS POLYSACCHARIDES, PEPTIDES, ALTERED LIGNINS THAT CAN BE SYNTHESIZED BY MICROORGANISMS OR CAN ARISE FROM MODIFICATIONS OF SIMILAR COMPOUNDS IN THE ORGANIC DEBRIS ARE NOT CONSIDERED TO BE HUMIC SUBSTANCES. The concept of Humic Substances as polymeric or macromolecular materials is now challenged and there is growing acceptance of pseudo-macromolecular assemblies arising from molecular associations.

4 HUMIN IS GENERALLY CONSIDERED TO BE A HUMIC COMPONENT IN INTIMATE ASSOCIATION WITH THE SOIL CLAYS. IT WILL BE SHOWN THAT SUCH MATERIALS DO NOT SATISFY THE CRITERIA OUTLINED IN THE CLASSICAL DEFINITIONS OF HUMIC SUBSTANCES

5 Any humic material recovered from soil by whatever means, after exhaustive extractions in aqueous base, can be considered to be Humin

6 Hydrogen Bonding and cation bridging are major mechanisms that inhibit the solvation of humic and other organic molecules in soils

7 Divalent and polyvalent cations can be displaced by H + -exchanging or by chelation. Hydrogen bonds can be broken by urea and by some organic solvents

8 Uses of Urea Urea (8M) is used in Biological Chemistry to break Hydrogen bonds. After exhaustive extractions of H + - exchanged soil, we use urea, in 0.1M NaOH. There were little differences between the amounts of additional organic materials isolated by 8M compared with 6M urea.

9 Urea (Continued) Optical density of Extractions Optical density Molarity of Urea 1st Extraction 6M 2nd Extraction 6M 1st Extraction 8M

10 Thus we use 6M urea in 0.1M NaOH in our exhaustive extraction procedures (following exhaustive extraction in 0.1 M NaOH)

11 In the classical definitions the material isolated in the 0.1M NaOH + 6M urea system described would be regarded as Humin

12 Principles Involved in Solvation When solvent molecules cluster around a solute molecule solvation takes place. Solvation is governed by properties of the solvent and the solute. The relative polarities of solvent and of solute are important in considerations of solubilities

13 Table 1 Relevant Properties of Organic Solvents

14 Table 2

15 Good Organic Solvents The Absorbance Values listed were compiled by multiplying the optical density observed by the dilution factor (using the solvent as diluent). Consider AVs of 18 and above as Good Solvents All these have molar volumes of 77 or less. All have viscosity values of 2 or less. All have Electrostatic Factor values of 140 or more. All have pk HB values >2 (a measure of abilities to break hydrogen bonds)

16 Good Organic Solvents (Continued) Thus DMF, Formamide, and DMSO are good solvents, But Solutes cannot be recovered from these solvents by freeze drying. Boiling points are too high for solvent evaporation to be effective. Resin technology can be used.

17 Again Any material that can be considered to be humic that is extracted in organic solvents, after exhaustive extractions in base, would, in the classical definitions, be HUMIN

18 We have seen that DMSO is the best of the organic solvents tested for the solvation of H + -exchanged humic acid

19 But, DMSO is a poor solvent for anions (though a good solvent for cations) Thus it is essential to have the soil/clay medium H + -exchanged in order to use DMSO.

20 DMSO plus concd H 2 SO 4 (94:6 in volume): An excellent solvent for aqueous base insoluble humin fractions DMSO is a excellent solvent to penetrate clay minerals A small amount of H 2 SO 4 protonates Ionized HS allowing Solvation in DMSO. High solvation (45-65%) takes place Exothermic reaction avoided for dry clay samples. Mild solvent for extraction of recalcitrant humin. No significant structural changes observed.

21 Flow chart for sequential exhaustive extraction of SOM

22 H + -exchanged soil Sequential exhaustive Extraction at ph 7, 10.6 and 12.6, under N 2 Alkaline extracts Dilute <20 ppm, adjust ph 2 XAD-8 Crude humin (soil residue) HAs at ph7, 10.6 and 12.6 XAD-4 FAs at ph7, 10.6, and 12.6 Insoluble humin XAD-4 acids 0.1M NaOH + 6 M Urea in N 2 Urea extracts Dry humic-rich clay DMSO + 6% H 2 SO 4 Dilute <20ppm, adjust ph 2.5 Insoluble DMSO extract XAD-8 10% HF H 2 O DMSO insoluble Humin Insoluble dialysis Soluble XAD-8 resin Urea HA XAD-4 Urea FAs DMSO Humin DMSO FAs Urea XAD-4 acids

23 In Summary Our approach isolates soil organic substances at different ph values giving fractionation on the basis of charge density differences. Then in the sequential extraction we use 0.1M NaOH + 6M urea. Then DMSO + 6% H2SO4 The Urea and DMSO isolates are fractionated into operationally defined humic, fulvic and XAD-4 fractions. The residual material is recovered by dissolving the clay in HF

24 13 C Chemical Shift (ppm) 13 C Chemical Shift (ppm) 13 C Chemical Shift (ppm)

25 Variable Amplitude Cross Polarization(VACP) 13 C NMR Solid-state 13 C nuclear magnetic resonances (NMR) experiments were carried out using a Varian Inova spectrometer at 13 C and 1 H frequencies of and MHz, respectively. Jackobsen 5 mm MAS double-resonance probe heads were employed. The Variable Amplitude Cross Polarisation with Total Sidebands Suppression technique (VACP-TOSS) was applied with a contact time of 1 ms, a spinning speed of 5 khz, acquisition times of 13 ms, recycle delays of 500 ms.

26 We now show VACP spectra for humic acids isolated from soil at different ph values ranging from ph 7 to 12.6, in 0.5 M NaOH and 0.1M NaOU + Urea. The sequential extraction process was used

27 Urea HA 163 urea HA 0.5M NaOH HA ph 12.6 HA ph 10.6 HA ph C C h e m ic a l s h ift (p p m ) VACP-TOSS 13 C NMR spectra: HAs sequentially/exhaustively extracted at different ph and with 0.1M NaOH + 6M urea from maize-amended soil

28 Note how the evidence for lignin residues (O-aromatic and methoxyl resonances) increase as the ph of the extractant increased. That means that lesser humified humic substances were isolated at the higher ph values. Again, note the similarities between the base and urea extracts.

29 And, What about the DMSO + H 2 SO 4 system? Is it likely to degrade the organic materials and give rise to artefacts? Our evidence, based on NMR spectra for lignin, suggests that structural alterations do not occur

30 Comparison of VACP 13 C NMR spectra of lignin, and Of Lignin after sequential treatment with 0.1M NaOH+6M urea and with DMSO+6%H 2 SO 4 (a) lignin base/urea treated DMSO+6%H 2 SO 4 treated (b) Cellulose CChemicalShift(ppm) FTIR spectra of lignin (a) and Cellulose (b). Cellulose (DMSO+6% H 2 SO 4 treated)

31 128 HAs 171 IHSS Mollisol FAs a * * 72 VACP DD ph 7 d VACP DD b , VACP DD ph 12.6 e VACP DD c VACP 0.1M NaOH + 6M urea f VACP DD DD C Chemical shift (ppm) C Chemical shift (ppm)

32 173 Humic acids 128 Fulvic acids d a ph 7 VACP DD * VACP b , DD VACP DD ph 12.6 e VACP DD c VACP 0.1M NaOH + 6M urea f VACP DD DD C Chemical shifts (ppm) C Chemical shift (ppm)

33 Based on the NMR and IR evidence shown, we are reasonably confident that the DMSO + H 2 SO 4 system does not give rise to artefacts in the systems we work with

34 Let us look now at DMSO Humin, i.e. materials isolated in DMSO + 6% H 2 SO 4 after the soil had been exhaustively extracted in the solvent sequence ending with 0.1M NaoH + 6M urea

35 33 30 DMSO Humin DMSO Fulvic Acids full vacp CSA filter full vacp DD 30 23,19,14 DD 153, VACP-TOSS spectra of DMSO humin (a) and DMSO Fulvic acids, an uncultivated grassland surface soil from Clonroche, Ireland.

36 (a) DMSO humin VACP-TOSS DD Oak Park 33 VACP-TOSS spectra (thin line) and corresponding DD spectra (thick line) of DMSO humin (a), DMSO insolulbe humin (b) and an expanded CSA filtered spectrum of DMSO insoluble humin (c) (c) DMSO insoluble humin. CSA-filtered , (b) DMSO insoluble humin VACP-TOSS DD a Oak Park Transmittance% b C Chemical shift (ppm) wavelength (cm -1 ) FT-IR spectra of DMSO humin (a), and DMSO insoluble humin (b).

37 VACP-TOSS DD VACP-TOSS DD a b C Chemical shift (ppm) 13 C C hem icalshift (ppm )

38 DMSO/H 2 SO 4 extracted significant amount of DMSO humin, and a small amount of water soluble DMSO fulvic- acids like fractions (<5%). DMSO humin is dominanted by highly aliphatic moieties, low aromatic carbon, and by strong contributions from polysaccharides,and polypeptides. CSA filtered spectra are well resolved, indicating strong resonances from anomeric C (carbohydrates), crystalline polymethylene aliphatic (33 ppm) and amorphous polymethylene aliphatic carbon from long chain alkanes, waxes, culticular material and/or long chain fatty acids, etc. DD spectra showed lower resonances from 55 ppm and in the O- aromatic region, suggesting low altered lignin residues, and the contribution from polypeptides and lignin or lignin derived mateiral. Compared with DMSO humin, DMSO fulvic acids are more oxidized, have more mobile polysaccharide-like structures.

39 Liquid State NMR of Humin Materials We now look at liquid state NMR spectra of Urea- and DMSO/H 2 SO 4 isolated Humin soil components

40 amide phenylalanine Aromatic in lignin Urea other protons in C DMSO * Anomeric protons in C α protons in P methoxyl in lignin P-OCO- (CH2) in LP N-acetyl group in PG -(CH2)n Urea humic acids, IHSS Mollisol -CH3 -γ(ch2) a Aliphatic C: carbohydrate P: polypeptides LP: lipoprotein PG: peptidoglycan DMSO humin, IHSS Mollisol b deuterium exchanged N-H to N-D due to addition of 4% D 2 SO 4 aromatic, amide carbohydrate, peptides, lignin, DMSO LP* PG ppm

41 ppm Total Correlation Spectroscopy (TOCSY) spectrum of DMSO humin isolated from DMSO+ 6% H 2 SO 4. General assignments are as follows: (1) Aliphatic Couplings; (2) Couplings from aliphatic alcohols and ethers - some amino acid side chains overlap in this region; (3) Couplings between α- protons and amino acid side chains in peptides/proteins. Couplings from ester will also overlap in this region; (4) Couplings from double bonds and, (5) Couplings from amide in peptides. Note these couplings are weak as most of the amides have been exchanged by the addition of D 2 SO ppm

42 ppm DMSO humin, IHSS Mollisol 2 PG 10 DMSO ppm Heteronuclear Multiple Quantum Coherence (HMQC) spectrum of the DMSO humin fraction, Mollisol IHSS standard. Assignments are as follows: (1) Aliphatic (for detailed assignment of this region; (2) R-OCO-CH 2 R methylene unit adjacent to the carbonyl in lipids (including lipoproteins and cutins); (3) CH 2 carbohydrate; (4) CH carbohydrate; (5) methoxyl; (6) α H-C in peptides/protein.

43 What can we Conclude? The procedures outlined show that, using sequential exhaustive extractions at increasing ph values, humic fractions can be isolated that are compositionally different, though still mixtures. Additional humic acids and fulvic acids are isolated in 0.1M NaOH + 6M urea systems. The mechanisms of release of these materials from the humin matrix may involve conformational alterations or cleavages of hydrogen bonding constraints. The residual materials released in DMSO + 6% H 2 SO 4 are compositionally different from the extracts in aqueous systems. These isolates would be classed as Humin Materials However, these do not satisfy the Operational definitions of Humic Substances

44 Conclusions (Continued) Thus, these so called humin materials are likely to be the most recalcitrant components of SOM. These are protected because of intimate associations between the molecules and clay surfaces. It is evident that, for the most part these are carbohydrates, peptides, peptidoglycans, waxes, lipids, aliphatic hydrocarbons, cutins, etc., but lignin derived (humic) subatances are likely to be there only because of entrapments. In other words the components in associations with the clays are predominantly organic molecules, whose structural types are well known.