JORNADA DEL AÑO INTERNACIONAL DE LOS SUELOS
EL IRNAS Y EL SUELO: Alianza y Complicidad
IRNAS‐CSIC, 30 Octubre 2015
Untie the knot of soil organic matter
chemistry enables additional
differentiation among soils and their
managements
Università di Napoli Federico II, Italy
Prof. Alessandro Piccolo
Centro di Ricerca Interdipartimentale sulla Risonanza
Magnetica Nucleare (NMR) per l'Ambiente, l'Agro-Alimentare
ed i Nuovi Materiali (CERMANU)
A little dialectic on
what is Humic Matter?
SUPRAMULECULAR
VS
MACROPOLYMERIC STRUCTURE
Humic substances (humic and fulvic acids) had been
traditionally described as macropolymers (10-500 KDa),
similarly to biopolymers, and their synthesis accounted to
extracellular enzymatic catalysis.
The assumed monomer of such a humic macropolymer has been
from time to time described with a hypothetical structure,
progressively updated according to the advance of analytical
techniques.
Stevenson, 1982
The macropolymeric theory
has never been
unambiguously proved by
direct experimental
evidence
Nevertheless, a number of
idealized structures, either
linear or branched, but all
stabilized by COVALENT
BONDS, became common in
textbooks and scientific
reports.
THE SUPRAMOLECOLAR STRUCTURE
HPSEC experiments indicated that conformations of humic substances
are altered by small amounts of organic acids.
These pioneered experiments undermined the macropolymeric theory
SIZE-EXCLUSION CHROMATOGRAPHY
CH3COOH
100
100
50
50
25
25
Void Volume
Retention Volume
Void Volume
Total Volume
Retention Volume
Piccolo et al., 1996. European Journal of Soil Science, 47:319-328;
Piccolo et al. 1999. European Journal of Soil Science, 50:687-694
Total Volume
HPSEC response to pH changes in solutions of true polymeric
standards such as polystirenesulphonates (PSS) and
polysaccharides (PYR)
UV detection
Refractive index detection
PSS
Piccolo, et al. Royal Society of Chemistry,
Special Publication no. 259, Cambridge, UK, (2000) pp.111-124
PYR
A reverse experiment: HA in different HPSEC eluents
A= NaNO3 pH 7; B= +CH3OH pH 6.9; C=+HCl pH 5.54; D=+CH3COOH pH 5.7
UV 280 nm
RI
D
D
Conte, Piccolo. Environmental Science & Technology (1999) 33:1682-1690
The Hypochromic effect due to
interactions of dipole moments of
chromophores with light.
A=control HA (0.05M NaCl) pH 7,
I=0.05
B=as in A but 4.6x10-7 M in CH3OH,
pH 6.97
C=as in A but to pH 5.54 with HCl
(<10-6M)
D=as in A but 4.6x10-7 M in
CH3COOH, pH 5.69
Piccolo (2001). Soil Science, 166:810-833
Polysaccharides
(PYR) standards
detected by RI
HPSEC response of true
polymeric standards eluted
by four different
modifications of eluents:
A= NaNO3 pH 7
B= +CH3OH pH 6.9
C=+HCl pH 5.54
D=+CH3COOH pH 5.7
Polystirenesulphonates
(PSS) standards
detected by UV
PSS-UV
Polystirenesulphonates
(PSS) standards
detected by RI
Piccolo. Soil Science (2001)
166:810-832
PYR-RI
MINIMIZATION OF CONFORMATIONAL ENERGY
POLYMERIC-COVALENT MODEL
Traditional humic
macropolymeric model
MW=6326 Da
Energy = 627.4 Kcal.mol-1
Conformational variation
induced by the adsorption
of 10 molecules of acetic
acid
Gain in
conformational
energy = 1.6 %
Energy = 617.26 Kcal.mol-1
HYPERCHEM software
PICCOLO et al. In: D.K. Benbi and R. Nieder, Editors. Handbook of processes and modelling in soil-plant system.
Haworth Press, New York, pp. 83-120 (2003).
MINIMIZATION OF CONFORMATIONAL ENERGY
SUPRAMOLECOLAR MODEL
HYPERCHEM software
Supramolecolar Humic
Model. Association of different
small molecules (aminoacid,
glucose, fatty acids, cinnamic
acids)
total MW = 3065 Da
Energy = 114.0 Kcal.mol-1
Gain in energy = 20.0 %
Conformational variation
induced by the approach of
10 molecules of acetic acid
Energy= 91.2 Kcal.mol-1
Gain in energy = 26.3 %
Conformational variation
induced by the adsorption of
10 molecules of acetic acid
Energy= 84.0 Kcal.mol-1
PICCOLO et al. In: D.K. Benbi and R. Nieder, Editors. Handbook of processes and modelling in soil-plant system.
Haworth Press, New York, pp. 83-120 (2003).
WHAT IS THE REAL MASS OF
HUMIC MOLECULES?
The evaluation of absolute molecular masses by ESI-MS
Electrospray ionization (ESI) is a soft technique to obtain
unfragmented and single-charged ions of humic molecules in
aqueous solution for compatible measurements by massspectrometry
Humic solutions are conveyed
through a capillary to the tip of a
cone where are ionized and
released in a droplet. The
droplets are progressively sizereduced by temperature until the
ions repel each other and enter
separated into the mass analyzer
in vacuum
ESI mass spectra (negative) of ammonium humates and fulvates
285.2
100
687
313
342
Volcanic HA
Mw < 1500 Da
694
369
875
1138
671
904
383
472
841
981
703
501
271
984
589
1202
1208
1363
1373
1510
1577
1749
1780
2338
1913
2046
2278
2408
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
m/z
100
271
284
Volcanic FA
Mw < 700 Da
3 44
433
438
489
521
558
59 1
693
794
9 16
980
1145
1251
14 20
1585
1757
1889
2018
2244
2412
0
200
400
600
8 00
1000
1200
1400
1600
1800
2000
2200
2400
m /z
709
100
626
601
781
444
429
489
880
393
908
1015
Lignite HA Mw
< 1400 Da
372
1109
305
1129
1235
298
293
1257
277
1400
1431
1722
1779
1926
2149
2268
2326
2411
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
m/z
Piccolo and Spiteller (2003). Analytical and Bioanalytical Chemistry, 377: 1047-1059
2400
No molecular mass larger than 1500 Da was noted by
FT-IRC-MS for terrrestrial humic matter
Ultra-high-resolution Mass Spectrometers (FT IRC-MS 11.7 Tesla) showed
that aquatic humic substances contain up to 6.000 different molecular
masses, while terrestrial humic sustances may contain more than 20.000
different masses
Bruker Daltonics, Bremen, D
Piccolo, Nebbioso and Spiteller (2010) Anal Bioanal Chem 397: 3071-3078
Diffusion Ordered NMR
SpectroscopY (DOSY-NMR)
One DOSY
Pulse
sequence
Pseudo 2D spectra
of diffusivity
(D=kT/6pr)
133 mg/mL
A nominal molecular weigh of
humic matter was calculated from
a DOSY calibration curve built with
standards of known MW
+ 5 mL
of Peat HA
of AcOH
5 mg/mL
of Peat HA
+ 5 mL
of AcOH
Alteration of the conformational
structure of humic matter by
addition of acetic acid was
visible also by DOSY
Calibration of Diffusion Coefficients (DC)
with standard compounds: glucose, sucrose,
raffinose, -cyclodextrin, maltodextrins, insulin
oxidized chain A, insulin dimer.
Simpson. Magnetic Resonance Chemistry, 2002, 40: S72–S82
THE NEW CONSENSUS
• Humic substances are composed of a multitude of
relatively small ( 1-1.5 kDa) and heterogeneous
molecules that self-assemble in supramolecular
associations
held
together
by weak
bonds
(hydrophobic and H bonds) in only apparently large
molecular sizes.
• The humic molecules are compounds surviving the
biochemical degradation of cellular components, they
randomly
associate
in
hydrophobic-hydrophilic
phases which are either contiguous to or contained in
each other. Their environmental reactivity is dictated
by their hydrophobic/hydrophilic ratio.
• The energetically fragile supramolecular associations
may be altered by interactions with organic acids and
metals that make bonds stronger than the hydrophobic
ones which stabilize the original humic conformation.
Piccolo. 2002. Advances in Agronomy, 75:57-134
Humic matter is a GENERIC MOLECULAR ENTITY that adjust
to different conditions, while cell biomolecules are SPECIFIC
since they are called to reproduce always the same
biochemical work
Modified after Hertkorn et
al. (2006) Geochim
Cosmochim Acta
INSIDE THE LIVING CELL
The genetic code controls the
synthesis of the discrete and
ripetitive molecules
OUTSIDE THE CELL
Casual Synthesis of humic
molecules, controlled only by
thermodynamics and kinetics within
the environmental boundaries
The complex heterogeneity of humic matter derives by its causual formation
without repetitive pathways
The difficulty in their analytical determination is due to its complex
supramolecular nature that inhibits reproducible identification of NOM
components
How do we exploit the supramolecular
theory to serve as an Episteme
(Science) for an innovative Techné
(Technology)?
A challenge for agricultural and environmental chemists is
to improve understanding of the structure and spacial
arrangement of organic molecules in soils and their
influence on plant nutrition
A detailed molecular characterization of soil organic matter
would allow a
Structure-Activity Relationship
between humic molecules and their activity on plants
To reach this goal, as for other field of Science
(Genomic, Proteomic, Metabonomic, Petroleomic),
HUMEOMICS
was introduced to enable a detailed structural mapping
of humic molecules
Humeomics
•The supramolecular concept implies that the small and
heterogeneous molecules forming the humic associations
may be selectively separated and analytically determined
one by one
•Humeomics is defined by a standardized procedure to
separate and characterize humic molecules
•Humeomics is possible due to the relatively weak forces
holding together humic molecules
•Humeomics implies no significant nor deliberate CarbonCarbon bond destruction
Nebbioso and Piccolo, Biomacromolecules 2011, 12, 1187–1199
Nebbioso and Piccolo, Analytica Chimica Acta, 2012, 720: 77– 90
Nebbioso et al. RSC Advance 2014. 4, 23658-65
Nebbioso et al. Biology and Fertility of Soils 2015, 51, 443–451
Nebbioso et al. Chemical and Biological Technologies in Agriculture 2014, 1:24
Humeomics consists into a sequential separation
of molecules and their analytical determination
1. Organic solvent extraction for molecules
structurally unbound to the humic matrix
2. Transesterification (BF3-MeOH) for molecules
weakly bound to the humic matrix through
ester bonds
3. Alkaline hydrolysis in MeOH for molecules
strongly bound to the humic matrix through
ester bonds
4. HI treatment for molecules strongly bound to
the humic matrix through ether bonds
The fractionation strategy
Acidic subfraction
HA from a soil (RES0)
Increasing
homogenity
Extract (ORG1) nonNeutral subfraction
covalent bound
Residue (RES1) Devoid
of unbound compounds
Acidic subfraction
Alcoholic
extract of loose
ester- bound
compounds
Org. Fr. (ORG2)
Neutral subfraction
Aq. Fr. (AQU2)
Residue (RES2) Devoid
of loose ester-bound
compounds
Residue (RES3) Devoid
Acidic subfraction
Alcoholic
extract of tight
ester- bound
compounds
Org. Fr. (ORG3)
Neutral subfraction
Aq. Fr. (AQU3)
O
O
BF3 12% inH OMeOH,
HO
90°C,
KOH
1M 8h
in MeOH,
of tight ester-bound
compounds
reflux, 2hH
I
HO
Extract (ORG4) Alkyl
iodides from ether cleavage
Residue (RES4) Devoid
of ether-bound compounds
HI 47% in H2O,
70°C, 48h
F
O
F
HO
O
HO
O
HO
OH
H
O
COOH
OH
O
F
OH
FO
OH
I
OH
+
O
O
B
B
R'
R
R'
R
O
O
O
CH2Cl2/MeOH;
RT;
HO
OO FH O HO
OO F
8h
OO
O
HO
R'R' H O
R'R'
RR OO
RR OO H
I H
+
I
F
B
O H
F R
R' O FO
OH
R
O
I
HO
COOH
HO
O
R
F
F
R'
B
R'
OH
OH
+ HO
O
O
OO F O H
O
HO
R'
RR OO
OO O H
R' O H
H
R
OH
OH
I
Humeomic fractionation
RES0
ORG1
ORG2
AQU2
ORG3
AQU3
ORG4
RES4
Applied Instrumental Methods
Solid Residues
13C-CPMAS-NMR
TMAH-Thermochemolysis-GC-MS
HPSEC-ESI-MS
Organosoluble fractions
1D- and 2D-NMR
GC-MS
1D- and 2D-NMR
Hydrosoluble fractions
TMAH-Thermochemolysis-GC-MS
HPSEC-ESI-MS
Nanjing 2012
Elaborating results: GC-MS, ESI-MS and NMR
74
74
100
O
O
87
O
O
87
87
%
55
69
0
83
67
97
87
115
107
129
127
M-43
M-31
143
157 171 185 199 213
147
167
187
207
227
M+
270
239
227
247
267
m/z
In GC-MS, fatty acids are derivatized and characterized by comparing their
fragmentation profile with a database
57
74
100
O
29
COSY experiments correlate protons
on adjacent carbons
O
TOCSY experiments correlate protons
on the same chain
87
%
M-57
55
69
97
0
46
66
199
143
86
115 129
106
126
157 171 185
146
166
186
213
206
H
M-29
256
227
226
M+
246
266
281
286
H
283.2619
C18H35O2
HSQC experiments show to which carbon
each proton is bound
Elettrospray source can characterize due to exact mass measurement.
However this route can not reveal isomerism
O
H
m/z
Likewise branched structures are revealed
283.1016
C10H19O9
H H
n
H H H H
OH
Humeomics on a HA extract
from a volcanic forest soil
GC-MS (TIC)
chromatograms
of:
(A) ORG1,
(B) ORG2
neutral
subfraction,
(C) ORG2
acidic
subfraction,
(D) ORG3
Nebbioso and Piccolo,
Biomacromolecules 2011,
12, 1187–1199
Amount (μg g-1 of original HA weight) of compound
classes detected by GC-MS in Humeomics fractions
from HA
Nebbioso and Piccolo, Biomacromolecules 2011, 12, 1187–1199
Nebbioso and Piccolo, Biomacromolecules 2011, 12, 1187–1199
HPSEC-ESI-MS chromatograms and mass spectra of RES0 and AQU2
HPSEC-ESI-MS (TIC) chromatogram of HA (RES0)
HPSEC-ESI-MS (TIC) chromatogram of AQU2
Amount (μg g-1 of original HA weight) of compound
classes detected by by HPSEC-ESI-MS in Humic
Fractions Separated by Humeomics
Nebbioso and Piccolo, Biomacromolecules 2011, 12, 1187–1199
1H-NMR
13C-CPMAS-NMR
1H-NMR
spectra
and 13C-CPMASNMR of humic
fractions separated
by Humeomics.
Nebbioso and Piccolo,
Biomacromolecules 2011, 12,
1187–1199
Weight yield of Humeomics on a HA extract
from a volcanic soil
% Yield of fractionation
100
80
Unaccounted weight = 40.20% due to losses of
inner water molecules, decarboxylation, and
volatile small compounds
60
40
20
0
RES0
ORG1
ORG2
AQU2
ORG3
Separated Fractions
Nebbioso and Piccolo, Biomacromolecules 2011, 12, 1187–1199
RES4
RES4 shows 66 % of quaternary sp2 carbon
Integral areas
Spectral
region
150-185
ppm
95-150
ppm
0-60
ppm
CPMAS
1.00
3.42
1.30
DD
1.00
2.47
0.42
2.47/3.42=~0.66
What are these quaternary
carbons?
Nebbioso and Piccolo,
Biomacromolecules 2011, 12, 1187–1199
Further HPSEC fractionation of RES4
f1
f10
f2
f3
f9
RES4
f4
f8
f5
f7
f6
Preparative HPSEC-UV chromatogram (280 nm)
of RES4: 5 mL of 0.2 mg/mL conc.
Gravimetric Yields
RES4f 1
2
3
4
5
6
7
8
9
10
Tot
Inj
mg
8.8
7.3
7.4
6.7
3.9
3.1
1.1
1.5
0.5
45
50
4.7
Nebbioso et al., 2014. RSC Advance 4, 23658-65
Empirical
formula
Size-Fractions
Bulk
RES4
1
2
3
4
5
6
Sum of
fractions
7
8
9
10
Alkanoic acids
C10H20O2
77
18
23
27
35
28
34
31
37
45
201
479
C12H24O2
48
27
29
35
47
33
83
54
40
61
103
512
C14H28O2
179
256
298
289
146
187
667
484
266
343
607
3544
C15H30O2
191
389
411
317
431
230
791
545
229
372
400
4113
C16H32O2
324
1000
1190
655
908
341
784
896
450
468
1090
7782
C17H34O2
25
144
242
98
132
57
179
143
53
96
134
1278
C18H36O2
8
122
145
104
422
25
67
160
50
59
99
1254
C14H26O2
46
37
52
64
80
36
168
106
53
98
117
812
C16H30O2
326
537
708
828
1032
518
2054
1290
496
1035
899
9397
C18H34O2
59
341
713
423
13108
168
507
415
150
678
625
Unsaturated alkanoic acids
Amount (µg g-1 of
total HA) of molecular
components
(empirical formulae)
found by hyphenated
HPSEC-ESI-MS for
RES4, ten sizefractions, and sum of
size-fractions, as
empirical formulae
17127
Unsaturated hydroxyacids
C6H10O3
33
15
24
24
38
58
34
65
32
46
176
511
C7H12O3
23
17
28
13
33
35
38
42
27
21
93
346
C18H34O3
22
46
21
50
72
29
49
41
18
106
243
675
Other unsaturated oxygenated acids
C15H22O4
9
22
24
12
73
67
186
368
71
217
426
1467
C17H26O4
13
60
21
44
37
41
59
450
163
154
111
1138
C17H26O5
19
12
9
6
40
36
65
233
59
103
80
642
C18H34O4
22
9
5
9
13
3
14
12
6
23
16
110
C18H36O4
4
33
8
8
43
2
7
5
3
22
27
157
C18H34O5
4
31
4
3
125
2
1
3
2
51
34
257
C19H30O5
2
28
10
8
10
19
23
391
9
103
12
611
C19H28O4N2
0
7
0
0
5
0
0
0
2
7
4
C19H30O6
8
7
7
2
23
25
54
256
26
125
52
576
C20H24O3
9
25
21
8
6
4
8
24
6
29
30
161
C21H26O3
8
19
16
3
4
2
5
12
3
15
16
94
C21H34O6
0
15
3
3
3
12
8
265
4
67
7
385
The sum of each
specific molecule in
the 10 fractions was
always
significantly
large that than found
in the orginal RES4
26
Cyclic acids
C7H6O2
4336
16336
17068
19570
10345
9587
12093
14422
47210
12619
34382
C6H4O5
7888
27484
9568
54330
28015
19636
23654
24775
55316
18970
53960
C7H6O8
9158
11842
9711
65224
16093
9249
9092
7834
8826
5427
3518
193632
315708
146816
Nebbioso et al., 2014. RSC
Advance 4, 23658-65
Structural characterization of some quaternary
carbons through HPSEC-ESI-MS/MS
Nebbioso et al., 2014. RSC Advance 4, 23658-65
DIRECT APPLICATION OF
HUMEOMICS TO AN
AGRICULTURAL SOIL
Quality and quantity of humic molecules obtained by Humeomics
directly applied on a agricultural soil (Torino silty-loamy) were
compared with those in a NOM extract from the same soil
SOIL
Sample
Unbound
NOM
Weakly
Ester
bound
Strongly
Ester
bound
Ether
bound
Mineral
bound
RES3
RES4
RES OM
ORG2
ORG3
AQU4
AQU2
AQU3
RES2
RES1
SOIL
ORG1
HCl
treatment
HF
treatment
Mass yield of OM extraction
2500
2000
1500
mg
1000
500
0
1250mg
2325mg
Percent yield of OM extraction
186%
2500
2000
1500
mg
1000
500
0
100%
C
H
N
163.9mg C
250
200
150
mg
100
50
0
Elemental Analysis
72.5mg C
The distribution of molecular
components in SOM by Humeomics
van Krevelen plots are built by Fourier Transform Ion
Cyclotron Resonance Mass Spectrometry (FTICR-MS)
Top-down approach by
FTICR-MS: get the
empirical formulae but not
the structure
van Krevelen Plot
Buttom-up approach by
Humeomics: get the
structural fomulae first and
then build a real van
Krevelen Plot
HPSEC-ESI-MS Orbitrap - for hydrosoluble AQU, RESOM and NOM
fractions
GC-MS - for organosoluble fractions (ORG)
NOM extract by ESI-MS Orbitrap
3.0
NOM
23 compounds Identified
Atomic ratio of H/C
2.5
2.0
1.5
1.0
0.5
0.0
0.5
1.0
1.5
Atomic ratio of O/C
45
NOM
35
30
25
20
15
10
5
0
AA
AC
AD
AL
AM
BA
DA
ES
ET
FA
HA
HN
HO
IM
KE
PA
PAH
PH
PE
RA
SA
SE
ST
SU
Relevant % Percentage
40
Humeomics
Unbound ORG1
Aliphatic C
 36% Fatty acids
 49% Alkanes
4000 3500 3000 2500 2000 1500 1000 cm-1
Unbound ORG1
HO
O
Unbound ORG1
3.0
ORG1
39 compounds Identified
Atomic ratio of H/C
2.5
2.0
1.5
1.0
0.5
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
0.5
1.0
1.5
Atomic ratio of O/C
ORG1
AA
AC
AD
AL
AM
BA
DA
ES
ET
FA
HA
HN
HO
IM
KE
PA
PAH
PH
PE
RA
SA
SE
ST
SU
Relevant % Percentage
0.0
Weakly Ester bound ORG2
C=O
Humeomics
C-O
 54% Fatty acids
 31% Sugars
4000 3500 3000 2500 2000 1500 1000 cm-1
Weakly Ester bound ORG2
OH
HO
HO
OH
O
OH
O
OH
HO
HO
OH
OH
HO
O
HO
O
Weakly Ester bound ORG2
3.0
ORG2
19 compounds Identified
Atomic ratio of H/C
2.5
2.0
1.5
1.0
0.5
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
0.5
1.0
1.5
Atomic ratio of O/C
ORG2
AA
AC
AD
AL
AM
BA
DA
ES
ET
FA
HA
HN
HO
IM
KE
PA
PAH
PH
PE
RA
SA
SE
ST
SU
Relevant % Percentage
0.0
Weakly Ester bound AQU2
 24% Benzoic
acids
 27% Amides
4000 3500 3000 2500 2000 1500 1000 cm-1
12
10
8
6
 H(ppm)
1
4
2
0
Weakly Ester bound AQU2
H3C
CH3
HN
H3C
H3C
O
OH
N
N
H
Weakly Ester bound AQU2
3.0
AQU2
33 compounds Identified
Atomic ratio of H/C
2.5
2.0
1.5
1.0
0.5
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
0.5
1.0
1.5
Atomic ratio of O/C
AQU2
AA
AC
AD
AL
AM
BA
DA
ES
ET
FA
HA
HN
HO
IM
KE
PA
PAH
PH
PE
RA
SA
SE
ST
SU
Relevant % Percentage
0.0
Strongly Ester bound ORG3
Humeomics
Aliphatic C
C=O
 43% Fatty acids
 33% Phenolic
acids
4000 3500 3000 2500 2000 1500 1000 cm-1
Strongly Ester bound ORG3
HO
O
HO
O
OH
HO
HO
O
O
OH
OH
Strongly Ester bound ORG3
3.0
ORG3
24 compounds Identified
Atomic ratio of H/C
2.5
2.0
1.5
1.0
0.5
0.0
0.5
1.0
1.5
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
ORG3
AA
AC
AD
AL
AM
BA
DA
ES
ET
FA
HA
HN
HO
IM
KE
PA
PAH
PH
PE
RA
SA
SE
ST
SU
Relevant % Percentage
Atomic ratio of O/C
Strongly Ester bound AQU3
 28% Esters
 15% Amines
4000 3500 3000 2500 2000 1500 1000 cm-1
Carbohydrates decreased
12
10
8
6
 H(ppm)
1
4
2
0
Strongly Ester bound AQU3
O
O
HO
NH
N
H
OH
NH2
CH3
CH3
O
O
H3C
CH3
H3C
O
OH
H3C
N
N
H
N
N
N
CH3
CH3
Strongly Ester bound AQU3
3.0
AQU3
40 compounds Identified
Atomic ratio of H/C
2.5
2.0
1.5
1.0
0.5
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
0.5
1.0
1.5
Atomic ratio of O/C
AQU3
AA
AC
AD
AL
AM
BA
DA
ES
ET
FA
HA
HN
HO
IM
KE
PA
PAH
PH
PE
RA
SA
SE
ST
SU
Relevant % Percentage
0.0
Ether bound AQU4
 47% Heterocyclic N
Humeomics
Ethers
 30% Amides
4000
3500
3000
2500
2000
1500
1000 cm-1
Ether bound AQU4
H3C
O
CH3
H
N
O
O
H3C
N
N
O
O
NH
H3C
O
NH
N
O
N
N
N
N
H3C
N
H3C
HO
O
CH3
Ether bound AQU4
3.0
AQU4
65 compounds Identified
Atomic ratio of H/C
2.5
2.0
1.5
1.0
0.5
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
0.5
1.0
1.5
Atomic ratio of O/C
AQU4
AA
AC
AD
AL
AM
BA
DA
ES
ET
FA
HA
HN
HO
IM
KE
PA
PAH
PH
PE
RA
SA
SE
ST
SU
Relevant % Percentage
0.0
Humeomics
Mineral bound RESOM
 14% Heterocyclic O
Aliphatic
HF treated
 66% Ethers
More Aromatic
NOM
4000
3500
3000
2500
2000
1500
1000 cm-1
RESOM
12
10
8
6
 H(ppm)
1
4
2
0
Mineral bound RESOM
OH
OH
HO
OH
HO
HO
OH
O
OH
O
HO
O
HO
O
O
HO
O
O
OH
O
O
O
O
H
HO
O
O
OH
O
OH
OH
HO
O
OH
Fe
O
O
O
Fe
Fe
O
Fe
Mineral bound RESOM
3.0
RESOM
31 compounds Identified
Atomic ratio of H/C
2.5
2.0
1.5
1.0
0.5
0.0
0.5
1.0
1.5
RESOM
AA
AC
AD
AL
AM
BA
DA
ES
ET
FA
HA
HN
HO
IM
KE
PA
PAH
PH
PE
RA
SA
SE
ST
SU
Relevant % Percentage
Atomic ratio of O/C
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
Humeomics improve detectability of
molecular components of SOM
ESI/MS on NOM extracted from Soil identified 23
compounds mainly aromatic esters.
ESI/MS & GC/MS on Humeomic fractions extracted
directly from Soil identified 249 compounds plus 29
isomers.
Humeomics improve detectability
3.0
3,0
AQU4
AQU2
ORG1
RESOM
AQU3
ORG2
ORG3
Atomic ratio of H/C
2.5
2,5
2.0
2,0
1.5
1,5
1.0
1,0
0.5
0,5
0.0
0,0
0.5
0,5
1.0
1,0
Atomic ratio of O/C
1.5
1,5
DIFFERENTIATING SOM
DYNAMICS IN AGRICULTURAL
SOIL BY HUMEOMICS
Soil under traditional tillage after
1 and 3 years
mg C
338.1mg C
350
300
300
250
256.1mg C
150.4mg C
250
149.8mg C
200
200
150
150
100
100
50
50
0
0
1st year
3rd year
AC, AD, KE
50
1,8
1,6
1,4
1,2
1,0
45
40
35
30
25
20
15
10
5
0
0,6
0,8
1,0
60
TRAIII ORG1
2,2
55
50
Relevant % Percentage
1,8
1,6
1,4
1,2
1,0
45
40
35
30
TRAIII ORG1
25
20
15
10
5
0
0,0
0,2
0,4
0,6
Atomic ratio of O/C
0,8
1,0
AC
AD
AH
AL
DA
BA
ES
FA
HA
HC
IM
KE
PA
PAH
PE
PH
RA
SA
SE
SES
ST
SU
Atomic ratio of H/C
2,0
Aliphatic Esters
0,4
Alkanes/alkenes
0,2
AC
AD
AH
AL
DA
BA
ES
FA
HA
HC
IM
KE
PA
PAH
PE
PH
RA
SA
SE
SES
ST
SU
0,0
Atomic ratio of O/C
0,8
TRAI ORG1
55
Relevant % Percentage
Atomic ratio of H/C
2,0
0,8
60
TRAI ORG1
2,2
60
TRAI ORG2
55
1,8
1,6
1,4
1,2
1,0
0,8
45
40
35
30
25
20
15
10
5
0,2
0,4
0,6
0,8
1,0
0
1,2
Atomic ratio of O/C
60
TRAIII ORG2
55
2,0
AC
AD
AH
AL
DA
BA
ES
FA
HA
HC
IM
KE
PA
PAH
PE
PH
RA
SA
SE
SES
ST
SU
0,0
TRAIII ORG2
50
1,8
Relevant % Percentage
1,6
1,4
1,2
1,0
0,8
45
40
35
30
25
20
15
10
5
0,0
0,2
0,4
0,6
0,8
Atomic ratio of O/C
1,0
1,2
0
AC
AD
AH
AL
DA
BA
ES
FA
HA
HC
IM
KE
PA
PAH
PE
PH
RA
SA
SE
SES
ST
SU
Atomic ratio of H/C
TRAI ORG2
50
Relevant % Percentage
Atomic ratio of H/C
2,0
2,2
60
TRAI ORG3
55
1,8
1,6
1,4
1,2
1,0
0,8
45
40
35
30
25
20
15
10
5
0,6
0,2
0,4
0,6
0,8
1,0
0
1,2
AC
AD
AH
AL
DA
BA
ES
FA
HA
HC
IM
KE
PA
PAH
PE
PH
RA
SA
SE
SES
ST
SU
0,0
Atomic ratio of O/C
2,2
60
TRAIII ORG3
55
2,0
50
1,8
Relevant % Percentage
1,6
1,4
1,2
1,0
0,8
0,6
45
TRAIII ORG3
C sequestration?
40
35
30
25
20
15
10
5
0,0
0,2
0,4
0,6
0,8
Atomic ratio of O/C
1,0
1,2
0
AC
AD
AH
AL
DA
BA
ES
FA
HA
HC
IM
KE
PA
PAH
PE
PH
RA
SA
SE
SES
ST
SU
Atomic ratio of H/C
TRAI ORG3
50
Relevant % Percentage
Atomic ratio of H/C
2,0
Fatty acids as a SOM marker
Originating from plant decay
TRA I
ORG1
ORG2
ORG3
TRA III
saturated FA
75%
82%
unsaturated FA
25%
18%
saturated FA
95%
90%
unsaturated FA
5%
10%
saturated FA
87%
95%
unsaturated FA
13%
5%
Originating from microbial activity possibly
because of the high sugar content of ORG2
Humeomics differentiate the
molecular composition of soils
and other biomasses
TERRA PRETA from Brazil
Labile SOM, possible auxin effect
mg C
800
More saccharides, possible
microbial activity enhancement
700
Oxisol Control soil
600
500
mg C
400
400
350
300
250
200
150
100
50
0
300
200
100
0
SEDIMENT from anoxic sea lagoon
(Greece)
More saccharides, possibly chitin derived
300
250
200
mg C 150
100
50
0
AQU1
ORG1
ORG2
AQU2
ORG3
AQU3
Natural Resins
mg C
mg C
80
80
60
60
50
50
40
40
30
20
65.6%
Loss
70
70
16.5%
Loss
30
20
10
10
0
0
Mastic (Chios, Greece)
Amber (Lettonia)
BIOCHAR from CHINA
mg C
Biochar
Humeomics
60
50
40
52%
Loss
30
20
10
0
mg C
Traditional NOM extraction
60
50
15%
Loss
40
Humeomics extracted 5 times more
identifiable carbon than the
alkaline extraction
30
20
10
0
starting
material
NOM
RES
material
Loss
Conclusions
Humeomics applied directly on agricultural soils provide the
molecular link between agricultural practices and SOM, and
carbon pools dynamics.
SOM evaluation by direct humeomics allow larger molecular
detectability than by NOM extraction.
Increased analytical yields
unfractionated humic matter
for
both
fractionated
and
Simplified readability of spectra and chromatograms of
humic matter after Humeomic fractionation
Unprecedented characterization of highly oxygenated cyclic
compounds containing non aromatic quaternary carbons
Significant enhancement of humic molecules separation
from soil
GENERAL CONCLUSIONS
Overall findings support understanding of humic matter as
SUPRAMOLECULAR ASSOCIATIONS of different classes of
molecules which are susceptible of structural identification
An improved and standardized of chemical fractionation, the
HUMEOMIC SCIENCE, may lead to a complete identification of the
molecular composition of humic superstructures.
Development of Humeomics may contribute to solve the StructureBioactivity relationship and cast the foundations of a modern
sustainable Agriculture
Advance knowledge of SOM molecular composition by Humeomics
may enable better understanding of soil processes and
technological control of organic carbon in soil (SOM dynamics
in different land use, OC sequestration in soil and its sink capacity,
fertility and soil quality maintanance).
THANKS TO YOU FOR ATTENTION
Special thanks for the essential collaboration to Dr Marios
Drosos and Dr. Antonio Nebbioso, post-docs at CERMANU
More information on www.suprahumic.unina.it
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