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Soil & Tillage Research 196 (2020) 104448

Contents lists available at ScienceDirect

Soil & Tillage Research


journal homepage: www.elsevier.com/locate/still

Molecular dynamics of organic matter in a tilled soil under short term wheat T
cultivation
Marios Drososa,b,*, Giovanni Vincib, Riccardo Spaccinib, Alessandro Piccolob,**
a
Institute of Resource, Ecosystem and Environment of Agriculture (IREEA), Nanjing Agricultural University, 1 Weigang Road, 210095, Nanjing, China
b
Centro Interdipartimentale di Ricerca sulla Risonanza Magnetica Nucleare per l’Ambiente, l’Agroalimentare ed i Nuovi Materiali (CERMANU), Università di Napoli
Federico II, via Università 100, 80055, Portici, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: Soil organic matter (SOM) or humus is essential for the agricultural and environmental functionality of soils.
SOM Humus comprises small-size heterogeneous organic molecules arranged in complex meta-stable suprastructures,
Humeomics whose composition can be greatly affected by land management. Here, we report the molecular dynamics of the
Wheat fractions extracted from an agricultural soil cropped with wheat after one and three years of tillage. The total
molecular components, named Humeome, of soil under wheat found in both hydrosoluble and organosoluble
fractions isolated by the Humeomic procedure, as determined by GC–MS and high-resolution Orbitrap LC–MS,
were compared to the Humeome characterized in the same soil when cropped with maize. While the three-years
tillage did not vary the total soil organic carbon under both wheat and maize, the carbon recovered for the sum
of Humeomic fractions isolated from soil was significantly larger for maize than for wheat, thus suggesting a
general destabilization of SOM under wheat cropping. Moreover, the soil Humeome under wheat resulted more
hydrophilic than under maize. While fatty acids and carbohydrates were periodically replenished by crop re-
sidues, nitrogen-containing molecules, such as amides and heterocyclic nitrogen, and iron-bound molecular
systems were the SOM components mostly reduced under wheat. The losses of these compound classes from the
soil Humeome was possibly attributed to the exudation differences between wheat and maize cropping. These
results reveal that the molecular dynamics and stability of SOM molecular components can be controlled by
crops even in a short term.

1. Introduction humic matter and plant species (Basler et al., 2015). While the concept
of ecological succession was introduced as a theory (Putnam, 1994),
World soils contain the greatest reservoir of organic carbon (OC) in few reports were so far published on the molecular changes of humus
the biosphere (Batjes, 2014), and innovative measures to reduce OC due to plant species, even though plant rhizodeposition is reckoned to
losses from soils are required to limit global changes (Minasny et al., play an important role in OC turnover in soil (Hütsch et al., 2002; Wang
2017; Piccolo et al., 2018). Nevertheless, agriculture intensification to et al., 2006). Larger microbial and CO2-C exudates were reported in
produce food for an increased population will inevitably put soil at risk wheat than in maize (Marx et al., 2007), while wheat residues were
of SOM degradation and fertility losses (Lal, 2009), thus menacing found to rapidly and persistently stimulate a microbial degradation of
human sustainability on the planet. Due to the still large uncertainty in fresh organic matter (Bernard et al., 2007), thereby enhancing miner-
strategies to sequester OC in soil (Baveye et al., 2018), there is an ur- alization of rhizospheric SOM (Schenck zu Schweinsberg-Mickan et al.,
gent need to enlarge knowledge on SOM dynamics, in order to control 2012). Though the long-term wheat cultivation was confirmed to ac-
soil OC and prevent degradation of soil fertility. celerate SOM transformation, heterocyclic nitrogen (HN) was identified
Humus represents the metabolic substrate for soil microbial activity by Fourier-transform infrared photoacoustic spectroscopy (FTIR-PAS)
that, in turn, continues to transform the bioavailable humic pools until as the most stable compound class (Du et al., 2014). Moreover, wheat
a new equilibrium is established between microbial communities, straw decomposition in soils was recently found to influence SOM


Corresponding author at: Institute of Resource, Ecosystem and Environment of Agriculture (IREEA), Nanjing Agricultural University, 1 Weigang Road, 210095,
Nanjing, China.
⁎⁎
Corresponding author at: Centro Interdipartimentale di Ricerca sulla Risonanza Magnetica Nucleare per l’Ambiente, l’Agroalimentare ed i Nuovi Materiali
(CERMANU), Università di Napoli Federico II, via Università 100, 80055, Portici, Italy.
E-mail addresses: drosos.marios@gmail.com (M. Drosos), alessandro.piccolo@unina.it, drososmarios@njau.edu.cn (A. Piccolo).

https://doi.org/10.1016/j.still.2019.104448
Received 3 January 2019; Received in revised form 3 October 2019; Accepted 8 October 2019
Available online 06 November 2019
0167-1987/ © 2019 Elsevier B.V. All rights reserved.
M. Drosos, et al. Soil & Tillage Research 196 (2020) 104448

molecular composition by enriching with alkyl C particulate and mi- BF3-MeOH (1 g of soil/1 mL of solution) and kept under N2 atmosphere
neral-associated OM (Chen et al., 2018). overnight at 85 °C. This transesterification was repeated twice, and the
The acknowledgment of the supramolecular nature of soil humus supernatants were centrifuged (15 min, 7000 rpm), and combined. The
(Piccolo, 2002, 2016) allowed to develop a novel SOM fractionation resulting solution was added with water to quench the residual BF3,
sequence, called Humeomics, that not only enabled separation of OC rotoevaporated to remove MeOH, and extracted three times with a total
from soil up to 3.6 times more efficiently than by traditional extraction of 150 mL of chloroform. The organic phase was separated (ORG2),
methods (Drosos and Piccolo, 2018), but also enhanced identification of dried with anhydrous Na2SO4, filtered on a Whatman 41 filter, and
single humic molecules by advanced analytical techniques, such as high rotoevaporated. The aqueous phase (AQU2) was rotoevaporated to re-
resolution ESI-Orbitrap and GC–MS (Drosos et al., 2017; Drosos and move residual MeOH and chloroform traces, and dialyzed against dis-
Piccolo, 2018; Drosos et al., 2018a). Humeomics progressively isolates tilled water using Amicon C membranes (1000 Da cutoff) until Cl-free,
molecules which are unbound to the humic suprastructure or released and freeze-dried. The remaining solid residue was air-dried.
from hydrolyzed esters and ethers (Nebbioso and Piccolo, 2011; Drosos The residue from ORG2 was suspended (w/v, 1 g/1 mL) in a 1 M
et al., 2018b), or from organomineral associations (Drosos et al., 2017; KOH solution in MeOH, and refluxed for 2 h at 70 °C under N2 atmo-
Drosos and Piccolo, 2018). This methodology was previously used to sphere. After cooling, the reaction mixture was centrifuged (10 min,
describe the soil Humeome (defined as the complete set of humic mo- 4500 rpm) and the supernatant recovered. The residue was washed
lecules in soil organic matter) present in a sandy loam soil under maize with 50 mL of MeOH and centrifuged. The supernatants were com-
cropping after one and three years of tillage (Drosos and Piccolo, 2018), bined, the pH adjusted to 2.0 with 37% HCl, and then liquid-liquid
whereas here we applied Humeomics on the same soil but under wheat extracted three times with a total of 150 mL (50:50, v/v) of DCM/water
cropping. The aim of this work was thus to verify whether wheat mixture. The organosoluble (ORG3) and hydrosoluble (AQU3) extracts
cropping may have resulted in a different molecular composition from were purified as for ORG2 and AQU2. The remaining solid residue was
that determined under maize for the same tillage time lapses. air-dried.
A suspension of 1 mL of 47% HI aqueous solution per g of soil re-
2. Materials and methods sidue from ORG3 was stirred for 48 h at 75 °C under N2 atmosphere.
After cooling, 100 mL of distilled water were added, stirred and filtered.
2.1. Soil The solution was neutralized by saturated NaHCO3 solution, freeze-
dried, and dialyzed (1000 Da cut-off membranes) first against a satu-
The Typic Ustifluvent soil (Eutric Fluvisol by FAO-Unesco) was rated Na2S2O3 solution to neutralize I2, and then, against distilled water
sampled from the experimental station of the University of Torino to remove residual Na2S2O3. The resulting suspension (AQU4) was
(44°53΄N, 7°41΄E, 232 m a.s.l) after one (2006) and three (2008) years freeze-dried.
of continuous conventional tillage. The soil had a sandy loam textural The residual soil was washed extensively with water and subjected
class (63% sand, 30% silt and 7% clay), pH 8, and a content of to extraction by alkaline solution (as for eSOM) of humic molecules
75 ppm K2O and 35 ppm P2O5. The soil had been fertilized with urea at tightly-bound to the soil inorganic matrix, representing a residual or-
the rate of 130 kg ha−1 of N and tilled by moldboard plowing 35 cm ganic matter (RESOM).
deep, followed by surface harrowing. Wheat (Triticum aestivum L., cv.
Blasco) was cropped in 10 m2 field plots in a completely randomized 2.4. eSOM and Humeomic fractions characterization
design of 3 plot replicates and 100 kg ha−1 of P2O5, and 200 kg ha−1 of
K2O per plot (Grignani et al., 2012). The organosoluble extracts (ORG1-3) were analysed by GC–MS,
while the molecular composition of hydrosoluble (AQU2-4), RESOM
2.2. Alkaline extractable SOM (eSOM) and eSOM was characterized by high resolution ESI-Orbitrap-MS. The
C, H, N content was determined by Elemental Analysis (Table 1), while
The eSOM fraction was extracted in triplicates from100 g of surface the iron content in soil was analysed by Atomic Adsorption Spectro-
soil (0–30 cm depth) using 0.9 L of an alkaline solution (0.5 M photometry.
NaOH + 0.1 M Na4P2O7). After overnight shaking, eSOM was separated
from soil by centrifugation (15 min, 4500 rpm), filtered through a 2.4.1. GC–MS
Whatman 41 filter, and adjusted to pH 7 with 37% HCl. The extract was Organosoluble fractions (ORG1-3) were methylated before GC–MS
dialyzed against distilled water using Amicon C membrane analysis using acetyl chloride/methanol, and then silylated using N,N-
(1000 Da cutoff) to remove residual salts and freeze-dried. The eSOM bis [trimethylsilyl] trifluoracetamide/1% trimethylchlorosilane.
extract amounted to 1086 ( ± 20) mg for the 1st year, and 1019 ( ± 30) Quantitative data were obtained adding nonadecanoic acid as internal
mg for the 3rd year (Table 1). standard, followed by an external calibration curve of specific standards
for the different classes of compounds. Methylated and silylated com-
2.3. Humeomics sequential fractionation pounds were converted to nominal masses by adding the H+ mass and
removing methyl and silyl groups, when needed. Chemical structures
Triplicates of 100 g of surface soil (0–30 cm depth) were placed in were obtained by NIST library. The peaks selected for identification
300 mL of 0.1 M HCl and shaken overnight. Each sample was cen- were the ones exceeding the cut-off limit of 0.05% of the overall
trifuged (15 min, 4500 rpm), and air-dried. The Humeomics fractiona- chromatographic area. Identified peaks were then recalculated to match
tion was applied as previously described (Drosos and Piccolo, 2018), in an overall relative percentage of 100.
order to obtain an unbound fraction (ORG1), weakly bound ester
fractions (ORG2 and AQU2), strongly bound ester fractions (ORG3 and 2.4.2. High resolution ESI-Orbitrap-MS
AQU3), strongly bound ether fraction (AQU4), and residual OM from Two milligrams of eSOM, hydrosoluble fractions (AQU2, AQU3 and
final soil (RESOM). AQU4) and RESOM were weighed, spiked with 20 μg each of the two
In particular, ORG1 was extracted under stirring for 24 h at room internal standards 16-d3-hexadecanoic acid and ring 13C labelled hy-
temperature from 100 g of washed soil suspended in 300 mL of a 2:1 v/ droxybenzoic acid, and then dissolved in vials using diluted ammonia
v dichloromethane (DCM) and methanol (MeOH) solution. The super- (0.05 M) LC–MS grade (Fluka) to reach a final volume of 1 mL. Samples
natant was separated by centrifugation (15 min, 7500 rpm) and filtra- were injected by an Agilent 1200 G1367 autosampler in two replicates
tion. The remaining soil residue was air-dried. using 40 μl of solution for each analysis. Mass spectra were obtained
The residue from ORG1 was placed in a Teflon tube added with 12% with an LTQ Orbitrap (Thermo Electron, Waltham, MA) equipped with

2
Table 1
M. Drosos, et al.

Mass yields, elemental analysis and percent visibility of organic matter of wheat cropped soil in eSOM and Humeomic fractions, as evaluated by referring to internal standards in GC–MS and ESI-Orbitrap-MS.
SAMPLE C% H% N% C/N ratio Mass yield (mg) C (mg)a H (mg)a N (mg)a OMtot OMvis (mg)c Visibility (%)d
(mg)b

Soil 1st year 1.05 ± 0.05 n.d. 0.08 ± 0.002 13.13 100000 1050 ± 50 n.d. 80 ± 2 n.d. n.d. n.d.
eSOM 17.7 ± 0.1 3.8 ± 0.1 1.9 ± 0.01 9.33 1086 ± 20 192.2 ± 3.5 41.3 ± 1.1 20.6 ± 0.1 455.46 184.01 40.4
RESe 0.49 ± 0.01 n.d. 0.03 ± 0.001 16.31 98000 ± 20 482.7 ± 9.8 n.d. 29.6 ± 1.0 n.d. n.d. n.d.
Loss of materialf 41.04 n.d. 3.26 12.59 914 ± 20 375.1 n.d. 29.8 n.d. n.d. n.d.
ORG1 28.3 ± 0.1 8.1 ± 0.1 0.7 ± 0.01 44.00 31 ± 1 8.8 ± 0.3 2.5 ± 0.3 0.2 ± 0.03 11.53 11.53 100
ORG2 34.2 ± 0.2 7.4 ± 0.1 1.1 ± 0.01 31.30 302 ± 15 103.3 ± 5.1 22.3 ± 2.6 3.3 ± 0.03 148.24 148.24 100
AQU2 17.4 ± 0.1 4.7 ± 0.1 2.5 ± 0.01 7.11 37 ± 2 6.4 ± 0.1 1.7 ± 0.1 0.9 ± 0.04 20.61 2.27 11
ORG3 22.7 ± 0.1 4.5 ± 0.1 0.8 ± 0.01 26.00 46 ± 2 10.4 ± 0.2 2.1 ± 0.1 0.4 ± 0.04 13.74 13.74 100
AQU3 9.9 ± 0.1 3.3 ± 0.1 1.4 ± 0.01 7.00 7±1 0.7 ± 0.01 0.2 ± 0.01 0.1 ± 0.01 n.d. n.d. n.d.
AQU4 1.6 ± 0.05 4.3 ± 0.1 0.2 ± 0.01 8.00 12300 ± 80 196.8 ± 6.0 528.9 ± 12.0 24.6 ± 1.2 627.00 469.62 74.9
RESOM 3.5 ± 0.1 2.5 ± 0.1 0.3 ± 0.01 11.77 1310 ± 20 45.9 ± 1.3 32.8 ± 1.0 3.9 ± 0.6 127.91 44.51 34.8
ORGsg 32.32 ± 1.5 7.10 ± 0.8 1.03 ± 0.3 31.41 379 ± 18 122.5 ± 5.6 26.9 ± 3.0 3.9 ± 1.0 173.51 173.51 100
RESOM &AQUsh 1.83 ± 0.05 4.13 ± 0.02 0.22 ± 0.02 8.47 13654 ± 103 249.8 ± 7.5 563.6 ± 3.0 29.5 ± 2.1 775.52 516.40 66.6
Total HUMEOMICSi 2.65 ± 0.09 4.21 ± 0.04 0.24 ± 0.02 11.15 14033 ± 121 372.3 ± 13.1 590.5 ± 6.0 33.4 ± 3.1 949.03 689.91 72.7
RESj 0.74 ± 0.01 n.d. 0.07 ± 0.01 10.60 65500 ± 125 484.7 ± 4.9 n.d. 45.9 ± 4.9 n.d. n.d. n.d.
Loss of materialk 0.94 n.d. 0.003 275.71 20467 ± 125 193.0 n.d. 0.7 n.d. n.d. n.d.
Soil 3rd year 1.11 ± 0.02 n.d. 0.08 ± 0.002 13.88 100000 1110.0 ± 30 n.d. 80 ± 2 n.d. n.d. n.d.
eSOM 17.0 ± 0.1 2.1 ± 0.1 1.6 ± 0.01 10.63 1019 ± 30 173.2 ± 3.0 21.4 ± 0.7 16.3 ± 0.3 399.87 167.14 41.8
RESe 0.38 ± 0.01 n.d. 0.06 ± 0.002 6.35 97800 ± 30 372.6 ± 9.8 n.d. 58.7 ± 2.0 n.d. n.d. n.d.
Loss of materialf 47.86 n.d. 0.42 113.04 1181 ± 30 565.2 n.d. 5.0 n.d. n.d. n.d.

3
ORG1 61.8 ± 0.2 6.5 ± 0.1 0.6 ± 0.01 49.50 16 ± 1 9.9 ± 0.6 1.0 ± 0.07 0.2 ± 0.01 12.91 10.51 81.4
ORG2 31.1 ± 0.1 0.6 ± 0.1 1.0 ± 0.01 31.09 330 ± 10 102.6 ± 3.1 2.0 ± 0.06 3.3 ± 0.1 152.44 43.60 28.6
AQU2 36.6 ± 0.2 3.1 ± 0.1 4.3 ± 0.01 8.36 32 ± 2 11.7 ± 0.7 1.0 ± 0.06 1.4 ± 0.09 21.55 5.62 26.1
ORG3 6.7 ± 0.2 1.1 ± 0.1 1.4 ± 0.01 12.33 110 ± 8 7.4 ± 0.5 1.2 ± 0.09 0.6 ± 0.11 9.89 1.69 17.1
AQU3 3.8 ± 0.1 2.0 ± 0.1 0.2 ± 0.01 4.75 50 ± 2 1.9 ± 0.1 1.0 ± 0.04 0.4 ± 0.01 n.d. n.d. n.d.
AQU4 1.6 ± 0.1 0.2 ± 0.1 0.7 ± 0.01 16.17 2326 ± 50 37.2 ± 0.8 4.7 ± 0.1 2.3 ± 0.35 106.69 106.69 100
RESOM 3.8 ± 0.1 0.2 ± 0.1 0.5 ± 0.01 12.55 660 ± 25 25.1 ± 1.0 1.0 ± 0.05 2.0 ± 0.13 55.07 16.91 30.7
ORGsg 26.29 ± 1.1 0.92 ± 0.1 0.90 ± 0.1 29.24 456 ± 19 119.9 ± 4.2 4.2 ± 0.22 4.1 ± 0.22 175.24 55.80 31.8
RESOM & AQUsh 2.47 ± 0.06 0.25 ± 0.01 0.20 ± 0.01 12.44 3068 ± 79 75.9 ± 2.6 7.7 ± 0.25 6.1 ± 0.58 183.31 129.22 70.5
Total HUMEOMICSi 5.6 ± 0.1 0.3 ± 0.05 0.3 ± 0.05 19.20 3524 ± 98 195.8 ± 6.8 11.9 ± 0.47 10.2 ± 0.8 358.55 185.02 51.6
RESj 0.36 ± 0.005 n.d. 0.04 ± 0.002 8.99 85400 ± 100 307.4 ± 0.4 n.d. 34.2 ± 0.04 n.d. n.d. n.d.
Loss of materialk 5.48 n.d. 2.91 17.04 11076 ± 100 606.8 n.d. 35.6 n.d. n.d. n.d.

a
CHN mass yields (mg) calculated by elemental analysis percentages multiplied by the mass yields (mg) of original materials and extracts. For soil, RES and loss of material H was not calculated by elemental analysis
and therefore mass yields were not defined (n.d.).
b
Chromatographic OMtot calculation is explained in Materials and Methods section 2.5.
c
Chromatographic OMvis calculation is based on internal standard.
d
Visibility (%) is based on the percentage of Chromatographic OMvis to the relative Chromatographic OMtot .
e
RES corresponds to residual material after eSOM extraction.
f
Loss of material after eSOM extraction based on original soil excluding eSOM CHN yields.
g
ORGs refer to the overall of ORG1, ORG2 and ORG3.
h
RESOM& AQUs refer to the overall of AQU2, AQU3, AQU4 and RESOM.
i
Total Humeomics refers to the addition of ORG1, ORG2, AQU2, ORG3, AQU3, AQU4 and RESOM fractions. Humeomics and eSOM extractions were conducted in triplicates. The values are referring to the average.
j
RES corresponds to residual material at the end of Humeomics after RESOM extraction.
k
Loss of material at the end of Humeomics after RESOM extraction based on original soil excluding Total Humeomics CHN yields.
Soil & Tillage Research 196 (2020) 104448
M. Drosos, et al. Soil & Tillage Research 196 (2020) 104448

a HESI-II source, using negative mode, 140–2000 m/z mass scan range, obtained:
and 1.0 s scan time. N2 was the sheath gas (50 AU) and He was the
(% OM ) × mi
collision gas (5 AU). Ion spray, capillary and tube lens voltages were set = mOMi
100
to 4000, 200 and 75 V, respectively. The ion source vaporizer and ca-
pillary temperatures were set to 350 and 275 °C, respectively. HPSEC The OM chromatographic visibility of each fraction (mOMi, vis ) for
comprised an Agilent 1200 G1312 Binary Pump set to output 0.5 mL both ESI-Orbitrap and GC measurements is calculated by multiplying
min−1 of a 55/45 A/B solution (A: 5 mM AcONH4 in Milli-Q water and mOMi with the percent visibility reported in Table 1:
5% MeCN, pH 7; B: 100% MeCN) for a total of 70 min in a Phenomenex mOMi × % visibility = mOMi, vis
Bio-Sep SEC-S 2000 column (300 × 7.8 mm) and precolumn
(30 × 7.8 mm), both thermostatted at 30 °C by an Agilent 1200 G1316 Finally, the total chromatographic OM can be calculated:
unit. UV chromatogram recordings were ensured by an Agilent 1200 n
G1315 DAD spectrophotometer set at 254 nm wavelength. The aver- ∑ mOMi = mOM1 + mOM 2 + …+mOMn
aged m/z values measured by Orbitrap MS were extracted from the i=1

Xcalibur software with C0-60 H0-120 O0-30 N0-10 Fe0-3 limits and 5 ppm as well as the total visible chromatographic OM:
mass tolerance, corrected on the basis of the internal standards, and
n
converted to nominal masses by adding the mass of H+ and removing
∑ mOMi,vis = mOM1,vis + mOM 2,vis + …+mOMn,vis
the masses of FeO, Fe2O2, or Fe3O3, when necessary. The most probable i=1
chemical structure for each empirical formula was found by the
ChemSpider (http://www.chemspider.com), and the PubChem
(https://pubchem.ncbi.nlm.nih.gov/) databases. As for GC–MS, the 3. Results and discussion
signals selected for identification were those exceeding the cut-off limit
of 0.05% of the overall area. Identified peaks were then recalculated to 3.1. Tillage effect on SOM dynamics of a wheat-cropped soil
match an overall relative percentage of 100.
The molecules composing eSOM and the other Humeomic fractions
2.4.3. Elemental analysis (Table S1) were identified by ESI-Orbitrap-MS and GC–MS, while their
Elemental Composition (C, H, N) of powdered samples (Table 1) analytical visibility was calculated, based on mass yields and elemental
was determined with a Fisons Instruments EA 1108 Elemental Analyzer, composition (Table 1).
using Eager 200 Ver. 3.09 calculation software. While only 30 mg OC per 100 g of soil were lost from the maize
cropped soil after 3 years of tillage (Drosos and Piccolo, 2018), the total
2.4.4. Atomic adsorption spectrophotometry soil OC under wheat was slightly, though insignificantly, enhanced by
A sample (2 g) of the soil prior to the extractions was suspended 60 mg per 100 g of soil at the 3rd tillage year, as revealed by elemental
overnight in 2 mL of HCl (37%) and then added to 48 mL of Milli-Q analysis (Table 1). Such addition of 3% OC per year due to wheat, is
water. The suspension was filtered through Whatman 41 filters and Fe similar to the 3% of SOC turnover per year in a surface soil under
in the filtered solution was measured by a Perkin Elmer AA700 Atomic Triticum aestivum earlier reported by Leavitt et al. (2001). Conversely,
Adsorption Spectrometer (AAS) in the graphite furnace mode. For both the eSOM extract from the 3rd year soil was 19 mg OC smaller than for
1st and 3rd year wheat cropped soil the Fe content was found to be the 1st year. Even larger was the OC reduction (47.4%) shown by the
2.37 ± 0.05%. sum of Humeomics fractions at the 3rd tillage year under wheat culti-
vation, being the AQU4 and RESOM fractions most responsible for this
2.5. Calculation of empirical formula loss (Table 1). This substantial OC decrease, estimated by elemental
analysis for the total Humeomics fractions, indicates that wheat crop-
A specific empirical formula CxHyOzNaFeb obtained from MS ping failed to stabilize small-sized labile molecules, probably added to
spectra, can be turned into its Formula Molecular Weight (FMW), soil by root exudation, which were then lost during the separation
FMW = 12x+1y+16z+14a+56b, where the atomic weight of each procedures.
element is multiplied by the number of corresponding atoms in each
compound. In the case of carbon atoms, the percent of total carbon 3.1.1. Single step alkaline extraction of SOM (eSOM)
(Ctot ) for all the identified compounds in each fraction is obtained by the The molecular composition of eSOM at the 1st year showed that the
following equation: most abundant compounds belonged to the heterocyclic nitrogen (HN)
n
class, followed by aliphatic ethers (ET), phenolic esters and/or ethers
12x i × (abundancei %)
Σ = Ctot (PE), and heterocyclic oxygen (HO). HN remained the main group in
i=1 100
the 3rd year, whereas ET, PE and HO were either degraded or lost, and
where (12x i ) and (abundancei %) are the total atomic weight and the amides (AD), amines (AM), dicarboxylic (DA), fatty (FA), and hydroxy
relative percentage of each ith molecule over the all visible compounds (HA) acids were extracted in larger yields than in the 1st year (Fig. 1).
in the mass spectrogram for every fraction. Concomitantly, the molecules found common in eSOM in both years
The total OM (OMtot ) for all the identified compounds in each showed a lesser abundance in the 3rd year (Fig. 2). These results sug-
fraction can be similarly calculated by taking into account the FMW of gest that wheat cropping should have induced a partial destabilization
each fraction (FMWi ) : of SOM hydrophobic components with tillage, thereby releasing polar
n compounds from their hydrophobic segregation (Spaccini et al., 2002)
FMWi × (abundancei %)
∑ 100
= OMtot and enabling their solubilization in the alkaline extraction medium.
i=1

Then, the percent of OM identified in each Humeomic fraction 3.1.2. Humeomics fractionation of SOM
(% OM ) can be calculated as follows: After the first tillage year, HN was confirmed, similarly to eSOM, as
the main component in the total Humeomics fractions, along with FA,
OMtot × Ci
= % OM DA, HA, AM, although benzoic (BA) and phenolic (PA) acids, phenols
Ctot
(PH), and sugars (SU) were additionally identified as significant classes
Where Ci is the percent carbon found in that fraction by elemental of compounds. However, these compounds were either less abundant or
analysis. The actual OM weight (mg) in each fraction (mOMi ) can then be depleted in the soil Humeome at the 3rd tillage year, with the exception

4
M. Drosos, et al. Soil & Tillage Research 196 (2020) 104448

Fig. 1. Weight distribution (mg OC) and Van Krevelen


Plots for compounds detected in eSOM, and Total
Humeomics after 1st (I) and 3rd (III) year wheat cul-
tivation. AA: Aminoacids, AC: Alcohols, AD: Amides,
AL: Alkanes/Alkenes/Alkynes, AM: Amines, BA:
Benzoic Acids, DA: Dicarboxylic Acids, ES: Aliphatic
Esters, ET: Ethers, FA: Fatty Acids, HA: Hydroxy Acids,
HC: Hydrocarbons not assigned elsewhere, HN:
Heterocyclic Nitrogen compounds, HO: Heterocyclic
Oxygen compounds, KE: Ketones, OS: Organic Sulfur
compounds, PA: Phenolic Acids, PAH: Polycyclic
Aromatic Hydrocarbons, PE: Phenolic Esters, PH:
Phenols, RA: Resin Acids, SA: Sugar Acids, SE:
Steroids, SES: Sugar Esters, ST: Sterols, SU: Sugars.
Standard deviation for all classes of compounds was
≤0.5 mg.

of FA and SU (Fig. 1). The more accurate molecular information ob- Humeome had been strongly bound to the soil matrix. The opposite
tained by Humeomics in respect to the traditional eSOM extract, in- trend was detected for the weakly ester-bound hydrosoluble (AQU2)
dicate that while SOM lost labile components under wheat cropping, and organosoluble (ORG2) fractions, for which the molecules common
the increased concentration of organosoluble/hydrophobic compounds to both years decreased with tillage time (Fig. 2). However, the OM
in soil was large enough to protect SOC from further degradation/loss, turnover in AQU2 favored less oxidized AM and FA over HN com-
and preserve, if not increase, the total OC in soil (Table 1). pounds (Fig. S3 and Table 2), while in ORG2 the new (III) molecules
The molecules identified in the unbound ORG1 fraction as common were placed in the more oxidized region of the van Krevelen plot
in both the 1st (I) and 3rd (III) year (Fig. 2), may have been transferred (Fig. 2).
with time from ORG2 and ORG3 fractions (Table S1). However, the The AQU4 fraction contained molecules mainly bound to iron hy-
more oxidized (I) molecules and alcohols (AC) (Fig. S1) were sub- droxides (Table S1). We showed earlier (Drosos and Piccolo, 2018) that
stituted in ORG 1 by FA (III) of larger molecular size (Fig. S2 and iron complexation to humic molecules is a major mechanism of SOM
Table 2). Similar trends were noted for the strongly ester-bound orga- stabilization. Here, the AQU4 of this study revealed the smallest degree
nosoluble fraction (ORG3) (Fig. 2), which lost PH (I) (Table 2) in favor of molecular turnover with time (Fig. 2), comprising only few sub-
of FA (III) (Figs. S1 and S2). Nevertheless, the molecules found common stitutions of AM molecules with HN compounds (Fig. S3 and S4). This
for both tillage years in ORG3 (and to a lesser extend in RESOM) raised behavior suggests again that the Humeome stability in AQU4 is attri-
in both percentage and concentration in the third year (Fig. 2 and Table butable to complexes with iron (Fig. 3). However, the covalent bonds of
S1), thus suggesting that some apolar esterified molecules in the Fe (C-O-Fe and C-N(H)-Fe) with humic molecules (Fig. S4), identified

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M. Drosos, et al. Soil & Tillage Research 196 (2020) 104448

Fig. 2. Van Krevelen Plots of eSOM and Humeomic fractions molecules and their relative OC percentages. Molecules present in both 1st and 3rd year samples are
noted in blue dots, their relative OC percentage in the 1st year is set as light blue bar (Common I) and as dark blue bar for the 3rd year (Common III). Molecules
present only in the first year are shown in green dots and their relative OC percentage as green bar (Only I), while molecules present only in the third year are shown
in red, and their relative OC percentage as red bar (Only III).

by Orbitrap MS, were not in the oxidized Fe3+, but in the reduced Fe2+ in AQU4 (I), thereby suggesting that such larger loss of AQU4 (III)
state, and in non-crystalline but amorphous structures (XRD data not material during dialysis than for maize (Drosos and Piccolo, 2018), may
shown). This should be due to the reducing properties of the soil Hu- have been probably due to the observed greater hydrophylicity of SOM
meome (Aeschbacher et al., 2012), since humic matter is well known to under wheat.
act as electron donor and capable to effectively reduce the native soil In RESOM, the maximum structural turnover found in the 3rd year,
Fe3+oxides in situ (Peretyazhko and Sposito, 2006). However, in the was attributed to the reduction of the molecular size (Fig. S4) of lipid
case of wheat, the molecules bound to iron in AQU4 (III) were less than molecules (Fig. 2), such as FA, DA, and AC, to the increase of reduced

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M. Drosos, et al. Soil & Tillage Research 196 (2020) 104448

Table 2
Compounds classes mg OC and (%) in eSOM and Humeomic fractions from soils after 1st and 3rd year of wheat cultivation.
1st year (I)

Sample

Group eSOM ORG1 ORG2 ORG3 AQU2 AQU4 RESOM Total ORGs Total AQUs &RESOM Humeomics
Totala

AA – – – – – – 0.18(0.4) – 0.18(0.07) 0.18(0.05)


AC – 3.67(41.7) 1.45(1.4) 0.08(0.8) – – 0.05(0.1) 5.20(4.23) 0.05(0.02) 5.25(1.41)
AD 0.38(0.2) – – – 0.05(0.7) 0.98(0.5) 0.50(1.1) – 1.53(0.62) 1.53(0.41)
AL – – 1.65(1.6) – – – – 1.65(1.34) – 1.65(0.44)
AM 1.35(0.7) – – – 0.01(0.2) 51.56(26.2) 0.28(0.6) – 51.85(20.85) 51.85(13.95)
BA 0.38(0.2) – – – – 7.08(3.6) traces(nn) – 7.08(2.85) 7.08(1.91)
DA 0.77(0.4) 0.03(0.4) 8.06(7.8) 0.07(0.7) – traces(nn) 0.09(0.2) 8.14(6.62) 0.09(0.04) 8.25(2.22)
ES 0.38(0.2) 0.11(1.2) 1.65(1.6) 0.05(0.5) 0.06(1.0) – 0.23(0.5) 1.87(1.52) 0.23(0.09) 2.10(0.57)
ET 29.03(15.1) – – – 0.38(5.9) 0.59(0.3) 3.21(7.0) – 4.18(1.68) 4.18(1.12)
FA unsat. – 1.05(11.9) 6.40(6.2) 0.53(5.1) 0.01(0.15) – 0.23(0.5) 7.98(6.5) 0.24(0.1) 8.22(2.21)
FA sat. – 2.70(30.7) 48.45(46.9) 4.60(44.0) 0.03(0.45) – 0.05(0.1) 55.75(45.37) 0.08(0.03) 55.83(15.02)
FA total – 3.75(42.6) 54.85(53.1) 5.13(49.1) 0.04(0.6) – 0.28(0.6) 63.73(51.87) 0.32(0.13) 64.05(17.23)
HA 0.19(0.1) – – 0.16(1.5) 0.03(0.4) 0.20(0.1) – 0.16(0.13) 0.23(0.09) 0.39(0.11)
HC – – – 0.03(0.3) – – – 0.03(0.02) – 0.03(0.01)
HN 120.72(62.8) 0.03(0.4) 1.55(1.5) 0.24(2.3) 5.21(81.1) 118.48(60.2) 38.09(83.1) 1.82(1.48) 161.78(65.04) 163.6(44.02)
HO 14.99(7.8) – – – 0.07(1.1) 1.18(0.6) 1.33(2.9) – 2.58(1.04) 2.58(0.69)
KE – – 0.83(0.8) – – – – 0.83(0.68) – 0.83(0.22)
OS – – – 0.06(0.6) – – – 0.06(0.05) – 0.06(0.02)
PA 0.96(0.5) 0.11(1.2) 5.68(5.5) 0.89(8.5) 0.02(0.3) traces(nn) 0.14(0.3) 6.68(5.44) 0.16(0.06) 6.84(1.84)
PAH – 0.03(0.4) – – – – – 0.03(0.02) – 0.03(0.01)
PE 19.61(10.2) 0.04(0.5) 0.52(0.5) 0.17(1.6) 0.03(0.4) 4.33(2.2) 0.60(1.3) 1.03(0.84) 4.66(1.87) 5.69(1.53)
PH 3.08(1.6) 0.08(0.9) 1.76(1.7) 2.9(27.8) 0.06(0.9) 12.0(6.1) 0.55(1.2) 4.74(3.86) 12.61(5.07) 17.35(4.67)
RA – 0.39(4.4) 3.5(3.4) 0.45(4.3) traces(nn) – – 4.34(3.53) traces(nn) 4.34(1.17)
SA – – – – – – – – – –
SE – – – – traces(nn) traces(nn) – – traces(nn) traces(nn)
SES – – – 0.05(0.5) 0.47(7.3) 0.4(0.2) – 0.05(0.04) 0.87(0.35) 0.92(0.25)
ST – 0.34(3.8) 1.76(1.7) – 0.01(0.1) – – 2.10(1.71) 0.01(nn) 2.11(0.57)
SU 0.38(0.2) 0.22(2.5) 20.04(19.4) 0.16(1.5) – – 0.32(0.7) 20.42(16.62) 0.32(0.13) 20.74(5.58)
3rd year (III)
AA 1.04(0.6) – 0.31(0.3) – – – 0.13(0.5) 0.31(0.26) 0.13(0.18) 0.44(0.23)
AC – 1.14(11.5) 2.26(2.2) 0.21(2.9) 0.02(0.2) – 2.51(10.0) 3.61(3.01) 2.53(3.42) 6.14(3.17)
AD 8.14(4.7) – 0.31(0.3) – 0.52(4.4) 1.3(3.5) 1.15(4.6) 0.31(0.26) 2.97(4.01) 3.28(1.69)
AL – 0.33(3.3) 0.72(0.7) 0.07(0.1) – – – 1.12(0.94) – 1.12(0.58)
AM 11.78(6.8) – 0.92(0.9) – 3.86(32.9) 1.12(3.0) 7.02(28.0) 0.92(0.77) 12(16.21) 12.92(6.66)
BA 1.04(0.6) – 0.72(0.7) – 0.39(3.3) 0.07(0.2) 0.5(2.0) 0.72(0.6) 0.96(1.3) 1.68(0.87)
DA 6.06(3.5) – 5.43(5.3) 0.04(0.6) 0.3(2.6) 0.07(0.2) 2.23(8.9) 5.47(4.56) 2.6(3.51) 8.07(4.16)
ES 1.39(0.8) 0.10(1.0) 5.43(5.3) 0.6(8.1) 0.32(2.7) – 0.03(0.1) 6.13(5.11) 0.35(0.47) 6.48(3.34)
ET 3.46(2.0) – 3.49(3.4) – 0.09(0.8) 0.04(0.1) 0.2(0.8) 3.49(2.91) 0.33(0.45) 3.82(1.97)
FA unsat. 5.72(3.3) 1.28(12.9) 0.83(0.8) 0.05(0.7) 0.25(2.1) – 0.67(2.7) 2.16(1.8) 0.92(1.24) 3.08(1.59)
FA sat. 9.70(5.6) 5.80(58.6) 39.70(38.7) 4.56(61.7) 1.94(16.6) – 2.64(10.5) 50.06(41.76) 4.58(6.19) 54.64(28.18)
FA total 15.42(8.9) 7.08(71.5) 40.53(39.5) 4.61(62.4) 2.19(18.7) – 3.31(13.2) 52.22(43.56) 5.50(7.43) 57.72(29.77)
HA 10.57(6.1) 0.64(6.5) 4.21(4.1) 0.12(1.6) 0.29(2.5) traces(nn) 0.48(1.9) 4.97(4.15) 0.77(1.04) 5.74(2.96)
HC – 0.15(1.5) 3.38(3.3) 0.44(6.0) – – – 3.97(3.31) – 3.97(2.05)
HN 107.06(61.8) 0.03(0.3) 3.38(3.3) 0.38(5.1) 2.6(22.2) 30.01(80.6) 4.63(18.5) 3.79(3.16) 37.24(50.32) 41.03(21.16)
HO 1.73(1.0) – – – 0.20(1.7) 0.07(0.2) 0.33(1.3) – 0.6(0.81) 0.60(0.31)
KE – – – – – – – – – –
OS – 0.10(1.0) – – – – – 0.1(0.08) – 0.10(0.05)
PA – 0.05(0.5) 6.99(6.8) 0.72(9.8) 0.23(2.0) 0.04(0.1) 0.05(0.2) 7.76(6.47) 0.32(0.43) 8.08(4.17)
PAH – – – – – – – – – –
PE 2.08(1.2) 0.03(0.4) – – 0.25(2.1) 0.22(0.6) 0.65(2.6) 0.03(0.03) 1.12(1.51) 1.15(0.59)
PH 0.69(0.4) – – – 0.15(1.3) 4.21(11.3) 1.05(4.2) – 5.41(7.31) 5.41(2.79)
RA – – – – – – 0.13(0.5) – 0.13(0.18) 0.13(0.07)
SA – – – – 0.2(1.7) – 0.28(1.1) – 0.48(0.65) 0.48(0.25)
SE traces(nn) – – – traces(nn) traces(nn) – – traces(nn) traces(nn)
SES – – 0.31(0.3) – 0.01(0.1) 0.07(0.2) – 0.31(0.26) 0.08(0.11) 0.39(0.2)
ST 0.17(0.1) 0.15(1.5) – – 0.09(0.8) – – 0.15(0.13) 0.09(0.12) 0.24(0.12)
SU 2.6(1.5) 0.1(1.0) 24.21(23.6) 0.18(2.5) – – 0.4(1.6) 24.49(20.43) 0.4(0.54) 24.89(12.84)

FA total are referring to the sum of the unsaturated (FA unsat.) and saturated (FA sat.) fatty acids.

AD compounds, and to the great losses of HN molecules (Fig. S3). 154.4 mg OC in eSOM (I) to 16.7 mg OC in RESOM (I) after Humeomics,
However, the presence of the same molecules in both eSOM and RESOM it signifies that they were the components mainly lost during dialysis of
(Table S1), suggests that some highly hydrophobic humic molecules the 1st year RESOM (Table 1). By excluding the OC of these molecules
could be released from the soil matrix only after an aqueous alkaline from the Humeomics losses, the unknown OC lost in the 1st year was
extraction. This implies that the 24 common molecules, mainly HN, had then adjusted to 55.4 mg, thus accounting only for 5.3% of the total OC.
not undergone any structural transformation during Humeomics, thus However, the 3rd year losses of SOC after Humeomics rose to 54.7%,
indicating that the sequential fractionation did not introduce extraction and remained as large as 48.3% even after exclusion of the OC for the
artifacts. Moreover, since these common molecules decreased from common molecules lost from RESOM (III). This observation reveals

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M. Drosos, et al. Soil & Tillage Research 196 (2020) 104448

Fig. 3. Van Krevelen plots of molecular structures in eSOM, AQU and RESOM fractions for both wheat cropped years. Molecules linked to iron hydroxides are shown
in red. AQU4 being the fraction with the highest mass of iron is more stable among the 1st and 3rd year. For AQU2, RESOM fractions and eSOM there are abundant
molecules in the 3rd year non bound to iron.

once more that the effect of tillage transformed SOM to less hydro- 3.2. Effects of wheat versus maize cropping on SOM dynamics
phobic and more labile structures, and that wheat cultivation magnifies
this process more than maize (Drosos and Piccolo, 2018). It has been earlier shown that traditional tillage affected the
Humeome of a soil cropped with maize over a 3 years period (Drosos
and Piccolo, 2018). Here, we applied Humeomics to the same soil and
tillage system but under wheat cropping, in order to show the

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M. Drosos, et al. Soil & Tillage Research 196 (2020) 104448

Fig. 4. Differences of the SOM main groups (mg


OC) extracted by Humeomics in total and identi-
fied by ESI-Orbitrap-MS (AQU2, AQU4, and
RESOM) and GC–MS (ORG1, ORG2, and ORG3),
for both Wheat and Maize crops after: A. one year
(I), and, B. three years (III) of cultivation. There
are 7 main classes of compounds: Heterocyclic
nitrogen compounds (HN), amides (AD), amines
(AM), fatty acids (FA), other lipidic dicarboxylic
(DA) and hydroxy (HA) acids, aromatic com-
pounds such as benzoic (BA) and phenolic (PA)
acids, phenolic esters (PE) and phenols (PH), and
sugars (SU).

differences on SOM dynamics due to the selected crop. After the 1st fucose, to the OC mg of plant-derived SU, such as glucose, xylose and
tillage year, the labile ORG1 fraction under wheat (Table 1, Table 2, arabinose (Table S1) (Larré-Larrouy et al., 2004; Basler et al., 2015; Lu
Table S1 and Fig. S1) showed a larger AL oxidation to AC than that et al., 2016), that changed from 0.76 (1st year wheat) to 0.81 (3rd year
found in ORG1 under maize (Drosos and Piccolo, 2018), while ORG2 wheat). However, such a minor variation was not reflected by the in-
lost 50% of abundance under wheat (Table 1) as compared to ORG2 variance in actinomycetes and total aerobic and anaerobic cellulolytic
under maize (Drosos and Piccolo, 2018), and ORG3 showed a larger bacterial populations observed in this soil with tillage (Ventorino et al.,
reduction of HA to FA, and of PA to PH under wheat (Table 1–2, Table 2012), although fungi were found significantly reduced in the 3rd til-
S1 and Fig. S1) than under maize (Drosos and Piccolo, 2018). lage year for both wheat and maize crops. The only significant differ-
In both wheat and maize cropping systems, the Humeome was ence between the two crops was in the 3rd year of cultivation, for which
composed by seven most abundant molecular classes (Fig. 4): three of more invertase activity was present under wheat than under maize
them represented nitrogen-containing compounds (HN, AD, AM), two (Ventorino et al., 2012). Larger levels of invertase may occur due to
were alkyl acids either as linear (FA) or functionalized (DA/HA) car- hydrolysis of sucrose to the more bioavailable glucose and fructose
boxylic acids, mainly present into organosoluble fractions, and the forms, probably related to the smaller content of sugar in SOM under
other two classes comprised hydrophobic aromatic molecules (BA/PA/ wheat than under maize (Fig. 4).
PE/PH), and a group of carbohydrate compounds (SU). These findings are in line with previous observations, which re-
While molecular transformation of these classes of compounds due ported that wheat straw decomposition enriched SOM with alkyl C
to tillage occurred in both cropping systems, there were specific (Chen et al., 2018). However, alkyl organic acids with more than one
changes directly linked to the cultivated crop (Fig. 4). The greatest functional groups (DA/HA) were found to be more abundant in the
difference was related to AD molecules, that were almost absent in the third than in the first year soil, regardless of the cropping system
case of wheat, whereas they represented not only the most abundant (Fig. 4). While AM and, mainly, HN molecules were more largely re-
group under maize, but also the most stable one (Drosos and Piccolo, presented in the first tillage year for wheat than for maize, they were
2018). In fact, AD molecules were already reported to be larger in soils lost after the third year more under wheat than under maize (Fig. 4).
cropped over a period of 6 years with maize than with wheat (Chen The same trend was observed for all abundant molecules in the Hu-
et al., 2017). While FA were largely abundant under maize after the first meome, except for the lipidic FA, DA and HA (Fig. 4), thus confirming
tillage year, but were greatly reduced after three years of tillage (Drosos previous findings which showed that SOC under wheat cropping be-
and Piccolo, 2018), their amount remained stable over three years came progressively more hydrophilic and labile (Shi et al., 2017).
under wheat cropping (Fig. 4). Similarly, soils cropped with wheat for Since the organic acid fraction of root exudates varies with crops
four years were earlier found rich in alkyl compounds, such as FA and (Hütsch et al., 2002; Wang et al., 2006), it is possible that wheat exu-
AL (Wiesenberg et al., 2010). dates may hydrolyze SOM more extensively than maize exudates. This
In our study, the FA molecules under wheat were progressively process may not only favor the release of labile hydrolyzed humic
degraded, while new plant-derived FA got replenished (Table 2 and molecules but also induce further SOM solubilization by displacing
Table S1). In fact, the C16:1 to C16:0 ratio raised from 0.014 to 0.048 humic molecules from complexes with iron (Drosos and Piccolo, 2018;
(Table 2) from 1st to 3rd year of cropping, thus revealing an increased Nuzzo et al., 2018). In fact, it was reported that more root-borne water
contribution of microbial derived organic matter. However, since the soluble OC is present in the vicinity of wheat roots (Merbach et al.,
ratio remained lower than 0.1 in both years, most FA must have ori- 1999), and that wheat during the germination exudes 10 times more
ginated from plants (Wiesenberg et al., 2010). In fact, the Humeome phytosiderophores than that required for Fe plant uptake, although the
results for wheat cropping showed that the ratio of unsaturated to sa- siderophores significantly decline after 5 weeks (Oburger et al., 2014).
turated FA (Table 2) dropped gradually from the labile ORG1 fraction Furthermore, rhizobacteria were found to enhance the phytosider-
(0.389 and 0.221 in ORG1 (I) and ORG1 (III), respectively) to the ophores (Richardson et al., 2009) deriving from tryptophan (an amino
strongly ester-bound ORG3 fraction (0.115 and 0.011 in ORG3 (I) and acid largely abundant in wheat germ), responsible for lateral roots
ORG3 (III), respectively), thus revealing a progressive enhancement of growth (Vacheron et al., 2013).
less labile, strongly bound molecules. This may be explained not only While it is known that siderophores are related to HN and AD mo-
with an increase of the hydrophobic protection from microbial de- lecules (Balado et al., 2015), we showed earlier that 5-methoxy-
gradation, but also with the less favorable conditions to microbial ac- tryptophan bound to Fe2+ (C12H14N2O3FeO) was the most abundant
tivity in PA and PH rich environments (Fig. S1). molecule of the HN group found in the Humeome of the soil studied
The slight increase of the microbial derived organic material from here (Drosos et al., 2017; Drosos and Piccolo, 2018). This iron-bound
the first to the third year is also shown by the ratio of the OC mg of molecular complex was similarly present in SOM after the 1st tillage
microbially-derived SU, such as mannose, galactose, rhamnose and year under both wheat and maize, and, while it remained constant in

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(Franzluebbers et al., 1994). Evidence for this phenomenon was re- Quaglietta Chiarandà, F., Amao, M., Lupo, F., Bochicchio, R., 2012. Field plots and
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fluence of land management (Piccolo et al., 2018), but also to its https://doi.org/10.1051/agro:2008044.
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Declaration of interests 1016/S0016-7061(03)00259-3.
Leavitt, S.W., Pendall, E., Paul, E.A., Brooks, T., Kimball, B.A., Pinter Jr., P.J., Johnson,
H.B., Matthias, A., Wall, G.W., LaMorte, R.L., 2001. Stable-carbon isotopes and soil
The authors declare that they have no known competing financial
organic carbon in wheat under CO2 enrichment. New Phytol. 150, 305–314. https://
interests or personal relationships that could have appeared to influ- doi.org/10.1046/j.1469-8137.2001.00113.x.
ence the work reported in this paper. Lu, C., Cao, Y., He, C., Bao, X., Fang, R., Wang, Y., Chen, X., Shi, Y., Li, Q., 2016. Effects of
elevated O3 and CO2 on the relative contribution of carbohydrates to soil organic
matter in an agricultural soil. Soil Till. Res. 159, 47–55. https://doi.org/10.1016/j.
Acknowledgements still.2016.02.001.
Luo, W., Wang, X., Sardans, J., Wang, Z., Dijkstra, F.A., Lü, X.-T., Peñuelas, J., Han, X.,
The authors are grateful to Prof. Paola Vitaglione for access to ESI- 2018. Higher capability of C3 than C4 plants to use nitrogen inferred from nitrogen
stable isotopes along an aridity gradient. Plant Soil 428, 93–103. https://doi.org/10.
Orbitrap-MS, to Mr. Franco Scognamiglio for assistance in metal ana- 1007/s11104-018-3661-2.
lyses and Mrs. Claudia Savarese for assistance in the extractions. Marx, M., Buegger, F., Gattinger, A., Zsolnay, Á., Munch, J.C., 2007. Determination of the
fate of 13C labelled maize and wheat exudates in an agricultural soil during a short-
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Appendix A. Supplementary data 2389.2007.00911.x.
Merbach, W., Mirus, E., Knof, G., Remus, R., Ruppel, S., Russow, R., Gransee, A., Schulze,
Supplementary material related to this article can be found, in the J., 1999. Release of carbon and nitrogen compounds by plant roots and their possible
ecological importance. J. Plant Nutr. Soil Sci. 162, 373–383. https://doi.org/10.
online version, at doi:https://doi.org/10.1016/j.still.2019.104448. 1002/(SICI)1522-2624(199908)162:4<373::AID-JPLN373>3.0.CO;2-#.
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