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/6pr) 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 NEW INTERDISCIPLINARY JOURNAL Open access NOW ACCEPTING SUBMISSIONS www.chembioagro.com Chemical and Biological Technologies for Agriculture is an international, interdisciplinary, peer-reviewed forum for the discussion of all fields of agricultural chemistry and related technologies. The scope of this journal includes chemical and biochemical processes aimed to increase agricultural production and food security, the evaluation of quality and origin of raw primary products and their transformation into foods and chemicals, as well as environmental monitoring and remediation. This journal presents the first opportunity to bring together researchers from a wide number of disciplines within the agricultural chemical and biological sciences, from both industry and academia. The principle aim of Chemical and Biological Technologies for Agriculture is to allow the exchange of the most advanced chemical and biochemical knowledge to develop technologies which address one of the most pressing challenges of our times - sustaining a growing world population. Editor in Chief : Alessandro Piccolo, Università di Napoli Federico II HTTP://WWW.CHEMBIOAGRO.COM See you in Napoli, Italy! The gulf of Naples The Faculty of Agriculture in the Royal Palace of Portici