Unveiling the molecular composition of the unextractable soil

Unveiling the molecular composition of the unextractable soil organic fraction
(Humin) by Humeomics
Antonio Nebbioso, Giovanni Vinci, Marios Drosos, Riccardo Spaccini, Alessandro Piccolo*
Centro Interdipartimentale di Risonanza Magnetica Nucleare per l'Ambiente, l'Agroalimentare e i
Nuovi Materiali (CERMANU)
Università di Napoli Federico II, Via Università 100, 80055 Portici Italy
*Corresponding Author: alessandro.piccolo@unina.it
Extended Materials and Methods
1. Soil and Humins
A detailed description of the soil sampling site, and soil properties are reported elsewhere
(Conte P, Spaccini R, Chiarella M, Piccolo A. 2003. Chemical properties of humic substances in soils
of an Italian volcanic system. Geoderma 117:243-250). The soil sample was first subjected to
extraction of Humic (HA) and Fulvic Acids (FA) by a NaOH and Na4P2O7 solution, as previously
described (Piccolo A. 1988. Characteristics of soil humic substances extracted with some organic and
inorganic solvents and purified by the HCl-HF treatment. Soil Sci 146:418-426.). The soil residue
containing the unextractable humin (HUM1) was washed extensively with distilled water until neutral
pH and freeze-dried. An aliquot of HUM1 was subsequently treated twice with a 10% HF and 5% HCl
v/v water solution, for 12 h under nitrogen, in order to disrupt and solubilize soil clays, and thus obtain
a clay-depleted soil humin (HUM2). HUM2 was dialyzed (3500 Da cut-off membranes) against
distilled water until chloride-free and freeze-dried.
2. Humeomic fractionation
A structurally unbound fraction (ORG1) was separated from humin by organic solvents
extraction (CH2Cl2:MeOH 2:1) at room temperature overnight under magnetic stirring and
subsequently centrifuged and filtered. The resulting residue (RES1) was suspended overnight with BF3MeOH at 90°C in tightly closed teflon containers to undergo an acidic methanolysis of ester bonds and
yield weakly ester-bound molecules in organosoluble (ORG2) and hydrosoluble (AQU2) fractions. The
residue (RES2) was then subjected to an alkaline methanolysis with a KOH–MeOH solution to break
more stable ester bonds and liberate strongly ester-bound molecules, which were distributed, in
organosoluble (ORG3) and hydrosoluble (AQU3) fractions. The residue (RES3) was then suspended in
aqueous HI (47%), in order to cleave alkyl–alkyl and alkyl–aryl ether bonds, including acetal and ketal
bonds. This last step dissolved strongly-bound ether molecules in an organosoluble fraction (ORG4),
and left a final solid residue (RES4). The organosoluble fractions (ORG1–3) were further separated by
aminopropyl solid phase extraction (SPE) to yield neutral and acidic subfractions, which were silylated
with bis[trimethylsilyl]trifluoracetamide/1% trimethylchlorosilane, and identified by GC–MS
(Nebbioso A, Piccolo A. 2011. Basis of a Humeomics science: chemical fractionation and molecular
characterization of humic biosuprastructures. Biomacromolecules 12:1187-1199). The hydrosoluble
fractions (AQU2) and solid residues (RES3–4) were dialyzed (3500 Da cut-off) against water and
freeze-dried, before undergoing thermochemolysis.
3. Gas chromatography–mass spectrometry (GC/MS) instrumental parameters
The injector was set at 250°C and a fused-silica capillary column (Restek Rtxc-5MS, 30 m
length ×0.25 mm I.D. ×0.25 m film thickness) was used under a helium flow rate of 1.6 mL/min .
Oven temperature was increased from 100 °C to 300 °C at 4 ◦C min−1 and kept constant for 20 min.
The mass spectrometer operated in full scan mode in m/z 50–600 range, with EI energy of 70 eV, and
cycle time of 1.0 s. Compounds identification was supported by the NIST-MS library of mass spectra.
Quantitative measurements by GC/MS and thermochemolysis were obtained through an external
calibration curve of known standards such as derivatized tridecanoic, ω-hydroxyhexadecanoic,
docosandioic acids, and sitosterol (Fiorentino G, Spaccini R, Piccolo A. 2006. Separation of molecular
constituents from a humic acid by solid-phase extraction following a transesterification reaction.
Talanta 68:1135–1142; Spaccini R, Piccolo A. 2009. Molecular characteristics of humic acids extracted
from compost at increasing maturity stages. Soil Biol Biochem 41:1164–1172).
4. NMR spectroscopy instrumental parameters
Samples were fitted in 4 mm zirconia rotors with Kel-F caps and spun at 13 000 ± 2 Hz. A
recycle time of 1.0 s, an acquisition time of 20 ms, and 1510 points were adopted for each spectrum.
The scan number ranged between 500 and 700. Data were processed with Mestre-C software,
and all FID spectra were transformed with 100 Hz line broadening exponential type filter function and
2k zero filling.
Table SM1 Main products of thermochemolysis of HUM1 and HUM2 (numbers correspond to signals in
pyrograms in Figure SM1).
No. Compound
No. Compound
23 Pyrazole
24 4,5-dihydro-3,4,5-trimethyl-1H-pyrazole
25 2,6-diethylpyrazine
26 Methylpyridine
27 Dimethylpyrimidindione
28 Dimethylindole
29 Methoxyindole
30 2-methyl-1H-isoindole-1,3-dione
31 N-(N-acetyl-L-leucyl) leucine, butyl ester
10 Dimethoxybenzene
32 Urea
11 Trimethoxybenzene
33 Tetraethylurea
12 Methyl benzoate
34 Tetramethyl 1,2-ethandiamine
13 Methoxybenzoic acid, methyl ester
35 Methylcarbamate
14 Naphthalene
36 Diethyl carbamic ac., methyl ester
15 Methylnaphthalene
37 2,5-dimethylbenzothiophene
16 Dimethylnaphthalene
38 Squalene
17 Furancarboxylic acid, propyl ester
39 3-ethyl-2,4,5-trimethyl-1H-pyrrole
18 Methylbenzofurane
40 Non Determined
19 4-methoxy-3-methylisobenzofuranone
Saturated monocarboxylic acids
20 1,5,5-trimethylimidazolidindione
Unsaturated monocarboxylic acids
21 Methylpyrrolidindione
▼ Dicarboxylic acids
22 1-(1-oxo-2-propenyl)-2-pyrrolidinone
Alkane/alkene pairs
Figure SM1. GC-MS pyrograms (TIC) of humeomic fractions: A. AQU2 from HUM1; B. AQU2 from
HUM2; C. RES3 residue from HUM1 before HI treatment; D. RES3 residue from HUM2 before HI
treatment; E. RES4 final residue after HI treatment from HUM1; F. RES4 final residue after HI
treatment from HUM2. Numbers refer to compounds listed in Table SM1
Figure SM2.
C-CPMAS-NMR spectra of residues from HUM1: A. original soil humin (RES0); B.
residue from HUM1 before HI treatment (RES3); C. final residue after HI treatment (RES4).
Figure SM3.
C-CPMAS-NMR spectra of residues from HUM2: A. original soil humin(RES0); B.
residue from HUM1 before HI treatment (RES3); C. final residue after HI treatment (RES4).