From supramolecular to molecular structure of …...From supramolecular to molecular structure of...

From supramolecular to molecular structure

of humic substances. Implications in carbon

sequestration and plant growth

Centro Interdipartimentale di Ricerca su NMR per l’Ambiente, l’Agroalimentare ed in Nuovi

Materiali (CERMANU) and Dipartimento DISSPAPA

VII Brazilian Meeting on Humic Substances, EBSH

Florianópolis 2007

30 October - 1 November 2007

Alessandro Piccolo

Università di Napoli Federico II

Humic substances (humic and fulvic acids) have been traditionallydescribed as macropolymers (10-500 KDa), similarly to biopolymers,and their synthesis was attributed to an extracellular enzymaticcatalysis.

The assumed monomer of such a humic macropolymer has beenfrom time to time described with a hypothetical structure accordingto the updating of new experimental results obtained withprogressively more advanced analytical techniques.

Stevenson, 1982

Such macropolymeric

theory of humic

substances became an

accepted paradigm,

despite the fact that it

has been never

unambiguously proved

by direct experimental

evidence.

A number of ideal

structures, either linear

or branched, but all

stabilized by covalent

bonds, became common

in textbooks and

scientific reports.

CH3COOH

Retention VolumeVoid VolumeTotal Volume

100

50

25

Void VolumeTotal VolumeRetention Volume

100

50

25

THE SUPRAMOLECOLAR STRUCTURE Experimental results (HPSEC, NMR, ESI-MS) obtained in Porticiindicate that humic substances, rather than being macropolymers,are supramolecular self-associations of heterogeneous andrelatively small molecules stabilized by weak bonds (H-bonds, vander Waals, p-p, CH-p), which may be disrupted by small amountsof organic acids.

SIZE-EXCLUSION CHROMATOGRAPHY

Monocarboxylic acids

A= HA (volcanic soil) pH 7 Mw=32961

B= to pH 3.5 with HCl

DMw=+13.7%

C=with HCOOH

DMw=-20.8%

D=with CH3COOH

DMw=-29.8%

E=with CH3CH2COOH

DMw=-5.7%

F= with CH3(CH2)2COOH

DMw=+2.3%

CH3COOH

HCOOH

HCl

Piccolo, Conte, Cozzolino (1999). European Journal of Soil Science, 50:687-694

HPSEC elution in NaNO3 0.05 M pH 7

A= HA (oxidized coal)

pH 7

Mw=9418

B=to pH 3.5 with HCl

DMw=-8.4%

C= to pH 3.5 with

methansulphonic acid,

DMw=-17.0%

D= to pH 3.5 with

benzensulphonic acid

DMw=-4.7%


Sulphonic acids

Cozzolino, Conte, Piccolo (2001)

Soil Biology and Biochemistry, 33:563-571

Dicarboxylic acids

A=HA (oxidized coal) pH 7

B= to pH 3.5 with HCl

DMw=-14.4 %

C=with HOOC-COOH , 2/0=0.029 DMw=-41.7 %

D=with HOOC-CH2-COOH2/0=35.2 DMw=-48.2 %

E=with HOOC-(CH2)2-COOH ,

2/0=815 DMw=-50.0 %

F=with HOOC-(CH2)3-COOH2/0=856 DMw=-72.2 %


Piccolo, Conte, Spaccini, Chiarella (2003).

Biology and Fertility of Soils, 37:255-259

Inte

nsi

ty (

mV

)

EFFECT OF ELUENT COMPOSITION ON

MOLECULAR SIZE DISTRIBUTION OF HA

INSTEAD OF LOWERING THE pH OF HUMIC

SOLUTIONS, WERE THE MOBILE PHASES TO BE

VARIED IN pH BUT NOT IN IONIC STRENGTH

SOLUTIONS OF HUMIC SUBSTANCES DISSOLVED

WITH CONTROL ELUENT AT pH 7 WERE INJECTED

INTO HPSEC AND ELUTED WITH MOBILE PHASES

MODIFIED BY ADDITION WITH METHANOL (pH 6.97),

HCl (pH 5.54) AND ACETIC ACID (pH 5.7)

HA (Lignite) in different HPSEC eluents

A= NaNO3 pH 7; B= +CH3OH pH 6.9; C=+HCl pH 5.54; D=+CH3COOH pH 5.7

Conte, Piccolo. Environmental Science & Technology (1999) 33:1682-1690

UV 280 nm RI

D

D

Hypochromic effect due to

interactions of dipole moments of

chromophores with ligth.

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

HPSEC BEHAVIOUR HUMIC SUBSTANCES VERSUS

UNDISPUTED POLYMERS

POLYMERS SUCH AS POLYSACCHARIDES AND POLYSTYRENESULPHONATES ARE COMMONLY USED

AS PROXY STANDARDS TO EVALUATE NOMINAL MOLECULAR SIZES OF HUMIC SUBSTANCES

HOWEVER, IF HUMIC SUBSTANCES ARE SUPRAMOLECULAR ASSOCIATIONS, THEY SHOULD HAVE A DIFFERENT HPSEC BEHAVIOUR THAN REAL COVALENT POLYMERS SUCH AS POLYSACCHARIDES

AND POLYSTYRENESULPHONATES

THE TWO CLASSES OF COMPOUNDS WERE COMPARED WHEN AQUEOUS SOLUTIONS WERE MODIFIED BY

ADDITION OF EITHER HCL OR ACETIC ACID

HPSEC response to pH changes in solutions of true polymeric

standards such as polystirenesulphonates (UV) and

polysaccharides (Refractive Index)

UV detection Refractive index detection

Piccolo et al. (2000). Royal Society of Chemistry,

Special Publication no. 259, Cambridge, UK, pp.111-124

Polysaccharides standards detected by RI

Polystirenesulphonates standards detected by UV

Polystirenesulphonates standards detected by RI

Piccolo et al.. (2001) Soil Science,166:174-185

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

PSS

PYR

EFFECT OF HYDRATION ON HUMIC

ASSOCIATIONS

UV and RI detected HPSEC

chromatograms of humic acids dissolved

and eluted in 8M Urea solution.

I = HA from an oxidized coal

II= HA from a lignite

III= HA from a Danish agricultural soil

Piccolo (2001). Soil Science 166:174-185

These results indicate that humic substances are supramolecular

associations of heterogeneous and relatively small molecules held

together by weak bonds (H-bonds, p-p, CH-p). Thermodynamic

reasons force the random self-assembled molecules in only

apparently large molecular sizes.

The disruption of supramolecular associations by low amounts

organic acids is due to the formation of H-bonds with

complementary humic sites. These H-bonds are stronger than the

hydrophobic bonds stabilizing humic conformations at neutral and

alkaline pHs.

Humic conformations in solution depend on both the acidity and

hydrophobicity of added organic compounds.

Traditional humic macropolymeric

modelMW=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 %

MINIMIZATION OF CONFORMATIONAL ENERGYPOLYMERIC-COVALENT MODEL

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).

Supramolecolar Humic Model. Association of

different small molecules (aminoacid, glucose, fatty

acids, cinnamic acids) total MW = 3065 Da


Conformational variation induced by the approach of 10 molecules of acetic acidEnergy= 91.2 Kcal.mol-1

Conformational variation induced by the adsorption of 10 molecules of acetic

acidEnergy= 84.0 Kcal.mol-1

Gain in energy = 20.0 %

Gain in energy = 26.3 %

MINIMIZATION OF CONFORMATIONAL ENERGYSUPRAMOLECOLAR MODEL

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).

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 mass-spectrometry

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 size-reduced by temperature until the ions repel each other and enter separated into the mass

analyzer in vacuum

Piccolo and Spiteller (2003). Analytical and Bioanalytical Chemistry, 377: 1047-1059

Volcanic HA

Mw < 1500 Da

Volcanic FA

Mw < 700 Da

Lignite HA

Mw < 1400 Da

ESI mass spectra (negative) of ammonium humates and fulvates

200 400 600 8 00 1000 1200 1400 1600 1800 2000 2200 2400

0

271

284

3 44

433

438

489

521

558

59 1

693

794

9 16

980 11451251

14 20 1585 175724121889 2018 2244

100

m /z

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400

m /z

0

100285.2

687

313

342

694 875

369 1138671

904

383841

472981703

501271

984589

1202

1208

1363

1373 1510

1577 17492338

1780 1913

2278 24082046

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400

m /z

0

100709

626

601

781444

429489

880

393

908

1015

3721109

305

1129

1235298

293

1257 1400

2771431

1722

1779 226819262149 2326

2411

Fourier Transform Ion Resonance Cyclotron Mass Spectrometry (FT IRC-MS) with a 11.7 Tesla Magnet

646.44123

647.51488

647.96965

648.47299

648.77336

649.46852

650.62670

651.31916

652.01595

652.41727

652.77309

647 648 649 650 651 652 653 m/z

0

2

4

6

5x10

Intens.

527.16343

527.31777

527.33855

527.43964

527.05 527.10 527.15 527.20 527.25 527.30 527.35 527.40 m/z

0

1

2

3

6x10

Intens.

527.16343

527.31777

527.33855

Bruker Daltonics, Bremen, D

651.31916648.47299

By new advanced high-resolution Mass Spectrometers (FT IRC-MS), it

was possible to ascertain not only that humic molecules are singly-charged

and have low molecular mass (1000 Da), but also that aquatic humic

substances contain up to 6.000 different molecular masses, while terrestrial

humic sustances may contain more than 10.000 different masses.

Advanced High-Resolution Mass Spectrometers (FT-IRC-MS),

High resolution mass-

spectrometry excluded

occurrence of multiple

charges and

fragmentation during

ESI-MS of humic matter. (Stenson et al., Analytical

Chemistry. 2002, 74:4397-09)

No molecular mass larger

than 1500 Da was noted

by FT-IRC-MS for a

terrrestrial HA (Piccolo and Spiteller,

unpublished)

Liquid-state NMR spectroscopy allows a semi-quantitative evaluation

of the molecular sizes of polydiserse substances by DOSY diffusion

spectra which correlate molecular size with molecular motions under 1H-NMR conditions.

Measurement of molecular masses by Diffusion NMR

Spectroscopy

D=kT/6pr

r=Hydrodynamic radius

Diffusion is closely related to molecular size through the Stokes-Einstein

equation

DOSY Pulse

sequence:

stegs1s

DOSY-NMR spectroscopy was applied to evaluate the molecular sizes

of humic substances on the basis of calibration curves made with

molecules of known MW.

Measurement of molecular masses by Diffusion NMR Spectroscopy

Simpson. Magnetic Resonance Chemistry, 2002, 40: S72–S82

Diffusion coefficients (D) of

humic matter fall within an

interval of MW <2000Da

Diffusion Order NMR

Spectra evaluate the

molecular size

distributions of humic

associations through

measurements of diffusion

coefficients (D).

The relative molecular

sizes of different

compound classes present

in humic matter may be

evaluated by

extrapolating the 1D

spectra associated to the

pseudo 2D DOSY-NMR

spectra

DOSY-NMR of humic and fulvic acids

Smejkalova and Piccolo (2007).

Environmental Science and

Technology, in press.

Changes of aggregation in 9

humic samples (pH 12.8) and

one PSS standard (6780 Da), as

a function of concentation, as

revealed by DOSY-NMR

Diffusion Coefficients (D) in

different spectral regions

Increase in concentration

produces a decrease of D as a

result of increase in molecular

size due to self-association of

humic molecules

Aggregation


Environmental Science and Technology,

in press.

Disaggregation

Disaggregation in 9 humic

samples and one PSS standard

(6780 Da) when acetic acid was

added to 10 mg.mL-1 solution

down to pH 3.6 as revealed by

DOSY-NMR Diffusion

Coefficients (D) in different

spectral regions.

The pH reduction of solutions

produced dramatic decrease in

molecular size following the

disrupture of humic

supramolecular structures


Environmental Science and Technology,

in press.

MICELLIZATION

The Critical Micelle

Concentration (CMC) of

humic matter can be

calculated by NMR

spectrocopy by either a

change in Diffusion

Coefficient (DOSY-NMR)

or in chemical shift of

selected signals (HR-1H-

NMR).

Out of 9 different humics,

only two HA showed a

CMC at 4 mg.mL-1, while

the rest revealed only

continuous association

increase

* *


Environmental Science and

Technology, in press.

Humic substances are composed of relatively small ( 1-1.5 kDa) and

heterogeneous molecules that self-assembled in supramolecular

associations of only apparently large molecular sizes. The associations are

stabilized by weak bonds (H-bonds, p-p, CH-p) and the hydrophobic effect

that enhances solvation entropy in water.

The humic molecules, carbonaceous compounds surviving the biochemical

degradation of cellular components, 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 metals and organic molecules that are stronger than

hydrophobic bonds, and stabilize the original humic conformation. Then,

the supramolecular structure changes conformational stability and energy

content.

THE NEW CONSENSUS

THE SUPRAMOLECULAR NATURE OF HUMIC

SUBSTANCES CAN BE EXPLOITED IN

DIFFERENT WAYS:

1. Separation in smaller-size humic associations by

preparative HPSEC in order to reach a better insight of

their molecular structure

Preparative HPSEC

elution of a humic acid

from lignite in NaCl

0.05 M, pH 7, before

and after addition of

acetic acid to pH 3.5

Piccolo et al., 2002. Environmental

Science and Technology, 36,76-84

Pyrolysis-GC-Mass Spectrometry

Piccolo et al., 2002. Environmental

Science and Technology, 36,76-84

1D-1H-NMR spectra of fractions separated by preparative HPSEC from a lignite

HA, before and after AcOH treament

No AcOH AcOH

13C-CPMAS spectra of size-

fractions separated by

preparative HPSEC from a

lignite HA

Conte, Spaccini, Smejkalova, Nebbioso, Piccolo.

2007. Chemosphere, 69:1032–1039.

Preparative HPSEC isolation of 11 size-fraction

separated from HA of a volcanic soil

28

13

49

70

39

29

Chemical Shift (ppm)

35 28

14

199

176

131

107

75

60

36

50 0100150200250 0100150200250

Bulk HA

HA1

HA2

HA3

HA4

HA5

HA6

HA7

HA8

HA9

HA10

HA11

0100150200250 01001502002500100150200250 0100150200250 5050

69

243

13C-CPMAS-NMR spectra of fractions separated by

preparative HPSEC from HA of a volcanic soil

Conte, Spaccini, Piccolo. 2006. Analytical

and Bioanalytical Chemistry 386: 382–390.



DIFFERENT WAYS:

2. To reach a better understanding of the detailed

molecular structure. A Humeomic Science

The supramolecular concept implies that the

small and heterogeneous molecules forming

the humic associations may be selectively

separated and analytically determined one by

one.

A challenge for agricultural and environmental chemists entails

a better understanding of the intrinsic composition of organic

molecules in soils and its influence on plant nutrition

A detailed molecular characterization of soil organic matter

would allow a

Structure-Activity

relationship between the humic molecules and their activity

inside the plant cells.

As for other field of Science (Genomic, Proteomic,

Metabonomic, Petroleomic) the link between structure of

humic molecules and biological activity may represent the new

field of HUMEOMIC

1. Molecules structurally unbound to the humic matrix by

organic solvent extraction

2. Molecules weakly bound to the humic matrix through

ester bonds by transesterification (BF3-MeOH)

3. Molecules strongly bound to the humic matrix through

ester bonds by alkaline hydrolysis in MeOH

4. Molecules strongly bound to the humic matrix through

ether bonds by HI treatment

Sequential Chemical Fractionationby progressively removing molecular classes

Unbound components

Humic Sample

CH2Cl2/CH3OH (2:1)

TransesterificationBF3/CH3OH 12%

Alkaline Hydrolyisis

KOH/CH3OH 1M

HI 47%

Solid residue

Extract in CHCl3 Aqueous phase

Weakly bound

componentsResiduo solido

Strongly bound

componentSolid Residue

Strongly bound

ComponentsSolid Residue

Sequential Fractionation

CPMAS 13C NMR

Thermochemolysis-GC/MS

ESI/MS

Extract in CH2Cl2 Aqueous phase

Extract in CHCl3

Humic Sample

CH2Cl2/CH3OH (2:1)


Alkaline Hydrolysis

KOH/CH3OH 1M

HI 47%

Solid Residue

Aqueous phase

Weakly bound

componentSolid Residue

Strongly bound


Aqueous phase

Strongly bound


Extract in CHCl3Unbound Components

Extract in CH2Cl2

Unbound Components Extract in CH2Cl2

Unbound components Extract in CHCl3 Extract in CH2Cl2

CH2Cl2/CH3CH(OH)CH3 (2:1)

Neutral Fraction

CH3CH2OCH2CH3/CH3COOH (98:2)

Acid Fraction

SPE

CH2Cl2/CH3CH(OH)CH3 (2:1)

Neutral Fraction

CH3CH2OCH2CH3/CH3COOH (98:2)

Acid Fraction

Gas-chromatography/mass-spectrometry

Deriv

atiz

atio

n

Thermochemolysis

GC/MS

HI 47%


Solid Residue

Solid residue

Aqueous phase

Alkaline hydrolysis

KOH/CH3OH 1M

Solid residue

Aqueous phase

Humic Sample

CH2Cl2/CH3OH (2:1)

Solid residueExtract of free

components

Extract in CHCl3

Strongly bound

component

Weakly bound

component

Extract inCH2Cl2

Strongly bound

component(ether bonds)

ESI/MS

The proposed sequential fractionation was applied to

the following samples:

1. HA. Humic acid extracted from a volcanic soil

2. HUM1. Humin remaining in soil after HA extraction

3. HUM2. Humin remaining in soil after treatment of HUM1

with a 10% HF solution

and

1. HAT. Titrated and quartz-filtered HA

2. F1. HPSEC separated high MW fraction of HAT

3. F2. HPSEC separated medium MW fraction of HAT

4. F3. HPSEC separated low MW fraction of HAT

0

20000

40000

60000

80000

100000

120000

140000

Free L ip ids

B F3-M eO H

K O H -M eO H

HA HUM1 HUM2

mg

.g-1

TO

C

Total yields of compounds by GC-MS

A. INITIAL BULK

B. RESIDUE >KOH-MeOH

C. RESIDUE >HI

300 200 100 0 -50 300 200 100 0 -100 300 200 100 0 -100

A

B

C

A

B

C

A

B

C

HA HUM1 HUM2

CPMAS-13C-NMR spectra of humic samples

HA HUM1 HUM2

>KOH

MeOH

>HI >KOH

MeOH

>HI >KOH

MeOH

>HI

60-70 80-87 30-40 80-83 45-50 56-60

Percent of extraction yields (w/w)

esadecanoic acid, methyl ester

12-metil-tetradecanoic acid, methyl ester

linoleic acid, methyl ester

13-metil-tetradecanoic acid, methyl ester

oleic acid, methyl ester

docosandioic acid, dimethyl ester

10,16-bis(trimethyilsiloxy)-esadecanoic acid, methyl ester

9,10,18-tris(trimethylsiloxy)-ottadecanoic acid, methyl ester

mandelic acid,

methyl ester

3,4-dimetoxy-cinnamic acid,

methyl ester

3,5-dimetoxy-benzoic acid, methyl ester

b-sitosterol, TMS ether

300 200 100 0 -100

Aqueous fraction >BF3-MeOH

Aqueous fraction >HI

The aqueous fractions resulting from the sequential

fractionation cannot be analyzed by GC-MS. However,

their chracterization may be achieved by NMR,

Thermochemolysis, and ESI-MS

ESI-MS spectra of humic fractions of HA

100

%

225213151

167

185

265

269 376367283

357289

327

407431

439447

466 527493 595543

1.46e5

Bulk HA

/

100

%

235191

151223

250

251401265

266376

294311339

413

421

445 563482 550

537

570598

660

6.06e4

Aqueous fraction > BF3-MeOH

0

100

%

217

213

205

403235 371253

259329291

295

407

425606439 548

462498 613649

645681

8.83e4

Solid Residue > KOH-MeOH

225 325 425 525 625 725 825 925 1025 1125 1225 1325 1425 m/z0

100

%

396219

217

163195

241 391353

263

311281

410429 574567

448 472 558493

519

609

591633

684760689 853

Solid Residue > HI

5.49e4

709(±30)

727(±8)

782(±1)

735(±22)

Mw

SIZE-FRACTIONS SEPARATION FROM HA BY PREPARATIVE

HPSEC

Fraction 1High Molecular Sizes

Fraction 2Intermediate Molecular Sizes

Fraction 3Low Molecular Sizes

Total Yields (mg g-1) of Organic Extracts by GC/MS

0

5

10

15

20

25

30

35

40

Unbound Components

Weakly-bound Components

Strongly-bound Components

mg g

-1

HA Fraction 1 Fraction 2 Fraction 3

Distribution (mg/g) of solid residues resulting from

various steps of sequential fractionation (w/w)

0

200

400

600

800

1000

Residue after extraction of free

components

Residue after transesterification and

methanolysis

Residue after HI treatment

mg g

-1

HA Fraction 1 Fraction 2 Fraction 3



DIFFERENT WAYS:

3. To clarify the elusive concept of humification

Mean Residence Time >1000 years

The process of humification is directly related to the stability of

soil aggregates and hydrophobicity of humic matter

Goebel, Bachmann, Woche, Fischer. 2005. Geoderma, 128:80– 93

D anish soil G erm an soil Italian soil

% 1

3C

-OC

fro

m M

aiz

e

0

5

10

15

20

H A1-1st

Alkaline Extraction

FA1-1st

Alkaline Extraction

H E-H ydrophobic Extraction (Acetone)

H A2-2nd

Alkaline Extraction

FA2-2nd

Alkaline Extraction

D-13C signature of Organic Matter

Incorporation of maize residue in soils for one year resulted into a

distribution of 13C-OC into different layers of humic matter that was

excluded from extraction by hydrophobic protection

Spaccini, Piccolo, Haberhauer, Gerzabek. 2000. European Journal of Soil Science, 51, 583-594

HA

1

HA

1 HA

1

FA

1 FA

1

FA

1

Ace

ton

e-H

B

Ace

ton

e-H

B

Ace

ton

e-H

B

HA

2

HA

2

FA

2

FA

2

FA

2

THE CONCEPT OF HUMIFICATION

Humification in soil can be considered as a two-step process of (1) biodegradation of dead-cells components

(2) self-aggregation of hydrophobic products resisting biodegradation.

In light of the supramolecular model, one needs not to invoke the

formation of new covalent bonds as a degree of humification. process that leads to the production of humus.

Humification is the progressive self-association of hydrophobic molecules that resist biodegradation during wet/dry cycles.

The suprastructures are thermodinamically separated by the water medium and adsorbed on the surfaces of soil minerals

and/or other pre-existing humic aggregates. The exclusion from water means exclusion from microbial degradation leading to

long-term persistence of humic matter in soil.



DIFFERENT WAYS:

4. turning humic superstructures into macromolecular

polymers with larger molecular masses

TURNING HUMIC SUPERSTRUCTURES INTO

MACROMOLECULAR POLYMERS

The loosely bound associations of humic substances may then

be stabilized by forming oligomers or polymers by oxidative

coupling reactions through a free radicals mechanism favoured

by catalysts

Horseradish peroxidase and H2O2 as an oxidant is known to

catalyze the oxidative coupling of a number of natural phenols

with formations of oligomers of varying MW.

Thus the formation of covalent C-O-C bonds among proper

humic molecules (phenols) may lead to a progressive

polymerization of humic matter and to a higher chemical

stabilization.

A HA from lignite at pH 4.7

treated with peroxidase and

H2O2 and followed by

HPSEC before and after

addition of CH3COOH to

pH 3.5

A

B

C

D

ENZYME CATALYSIS

WITH H2O2 AS OXIDANT

Cozzolino and Piccolo. 2002. Organic

Geochemistry, 33, 281-294

Biomimetic catalysts

work as well as enzymes

in oxidative couplings

and are more

environmentally resistant

BIOMIMETIC

CATALYSIS WITH

H2O2 AS OXIDANT

A synthetic water-soluble

meso-tetra-(2,6-dichloro-

3-sulphonatophenyl)

porphyrinate of Fe (III)

chloride (FeP) was

employed as catalyst

Piccolo et al., 2005.

Biomacromolecules, 6: 351-358

OLIGOMERIZATION OF PHENOLIC HUMIC PRECURSORS

10 20 30 40 50

0

10

20

30

40

50

60

70

80

10 20 30 40 50

0

20

40

60

80

100

120

140

VI

V

IV

acid

acidcaffeic

p-coumaric

catechol

Inte

nsity

of f

luor

esce

nce

(mV)

Elution time (min)

A

B

III

II

I

acidacid

p-coumaric caffeic

catechol

Inte

nsity

of a

bsor

banc

e (m

V)

Elution time (min)

A

B

A. Substrate+H2O2

B. S + FeP + H2O2

Smejkalova and Piccolo. Environmental Science and Technology, 40, 1644-1649 (2006).

-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0 100 200 300 400 500 600

Time (min)

ln(c

t /c

0)

A

B

C

D

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0 100 200 300 400 500 600

Time (min)

ln(c

. t./

c0)

A

B

C

D

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0 100 200 300 400 500 600

Time (min)

ln(c

.t./c

0)

A

B

C

DA. (S);

B. (S+ H2O2);

C. (S+FeP);

D. (S+FeP+H2O2).

catechol caffeic acid

p-coumaric acid

RATES OF OLIGOMERIZATION

Smejkalova and Piccolo. Environmental

Science and Technology, 40, 1644-1649 (2006).

DD

D

C

C

C

RADICAL SPECIES RESPONSIBLE OF OXIDATIVE

OLIGOMERIZATION CATALYZED BY THE

BIOMIMETIC CATALYST

Smejkalova and Piccolo. Environmental Science and Technology, 40, 1644-1649 (2006).

OXIDATIVE

OLIGOMERS (C-C AND

C-O-C COUPLING)

IDENTIFIED BY

GC-MS

AFTER ISOLATION OF

PRODUCTS BY HPLC

AND SYLILATION

Smejkalova, Piccolo, Spiteller. Environmental


T

T = TRIMERS

D D

D

D D

D D

D D

D = DIMERS

T

OXIDATIVE OLIGOMERS

(C-C AND C-O-C

COUPLING)

IDENTIFIED BY

ESI-MS/MS

AFTER ISOLATION OF

PRODUCTS BY HPLC

Smejkalova, Piccolo, Spiteller. Environmental


D

T

TET

D

D

TD

TD

TET = TETRAMERS

D = DIMERS

T = TRIMERS

A= HA control (pH 7); B= HA + AcOH to pH 3.5

C = polymerized (pH7); D = as in C + AcOH to pH 3.5

HA from Lignite eluted in

phosphate buffer at pH 7

B

D

D

C




C = polymerized (pH7); D = as C + AcOH to pH 3.5

HA from oxidized carbon

B

D

D

C

C

B

Piccolo et al., 2005. Biomacromolecules, 6: 351-358


C = polymerized (pH7); D = as C + AcOH to pH 3.5

HA from volcanic soil

B+D

B+D

C

C

D



after treatment

before treatment

DRIFT SPECTRA OF HA FROM

AN OXIDIZED COAL 1590

1395

1046

1601

1386

1241

1094

1048

1500 1000

Wavenumber (cm-1)



H A1

H A2

H A3

TH

) (

)1

ms

T1H VALUES (ms) FROM CPMAS-NMR SPECTRA FOR

DIFFERENT HUMIC ACIDS INDICATE REDUCED

MOLECULAR MOTION AFTER A POLYMERIZATION

TREATMENT UNDER BIOMIMETIC CATALYSIS



Samplea Treatment g DH (mT) Spin g-1

(x1017)

P1/2 (max)

HA1 Control 2.0038 0.56+0.007 4.22+0.182 7.01

Polymerized 2.0036 0.62+0.003 4.32+0.106 1.47

HA2 Control 2.0036 0.53+0.000 1.29+0.0625 8.16

Polymerized 2.0036 0.55+0.000 2.15+0.0453 1.43

HA3 Control 2.0036 0.49+0.004 1.90+0.0185 6.50

Polymerized 2.0036 0.53+0.008 2.72+0.0565 1.49

Electron Spin Resonance values for g parameter, line width (DH), spin

concentration, and microwave power at half-saturation (P1/2) of humic samples

before (control) and after the polymerisation reaction

Piccolo et al., 2005. Biomacromolecules, 6: 351-358

The catalytic action of metal-porphirins in

polymerization of humic matter can be also

exerted as photosensitivization by the solar

electromagnetic radiation

Elution Volum e (m L)

10 15 20 25 30

Inte

ns

ity

(m

V)

0

20

40

60

80

100

120

140

160

180

H AL + FeP

H AL + FeP 13h irradiation

H AL + FeP 13h irradiation after 8 days


Mw

(D

a)

0

1000

2000

3000

4000

5000

6000

7000

U V D etector (280 nm )

Smejkalova and Piccolo et al., 2005. Biomacromolecules, 6: 2120-2125

Elution Volum e (m L)

10 15 20 25 30

Inte

ns

ity

(m

V)

10

12

14

16

18

20

H AL + FeP

H AL + FeP 13h irradiation



Mw

(D

a)

0

2000

4000

6000

8000

10000

12000

14000

W hole chrom atogram

1st peak

R efractive Index D etector

Smejkalova and Piccolo et al., 2005. Biomacromolecules, 6: 2120-2125



DIFFERENT WAYS:

5. To sequester organic carbon in soil

20 mL water

40g Fluventic Xerochrept (2 mm)

2 mg FePha

(10 kg.ha-1)

3 mL H2O2

(8.6 M)

5 days incubation

room temperature

1. Separation of water

stable aggregates

2. Particle-size

fractionation

3. OC determination by

elemental analyser

4. Alkaline extraction of

humic and fulvic acids

5. Determination of CO2

emission by respiration

EXPERIMENTAL

Control

Treated with H2O2

Added with Catalyst and H2O2

Mean Weight Diameter in water

0

5

10

15

20

25

30

35

a a

b

An index of structural stability

Control

Treated with H2O2

Added with FePha and H2O2

0

2

4

6

8

10

12

14

16

18

20

4.0-2.0 2.0-1.0 1.0-0.5 0.5-0.25 <0.25

Aggregate size (mm)

Yie

ld (

g)

0

5

10

15

20

25

30

35

40

Control Added with H2O2 Treated with FePha

and H2O2

Yie

ld (

g)

Water stable aggregates

Macroaggregates

Microaggregates

>250 mm

<250 mm

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

HA FA

Yie

ld (

%)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

HA FA

Yie

ld (

%)

40g 200g

Control

Treated with FePha and H2O2

Extraction yields (%) of HA and FA

from 40 g and 200 g of soil

Organic carbon distribution in particle-size fractions

as compared to bulk soil

0

4

8

12

16

20

Org

an

ic C

arb

on

(g

.kg

)-1

A B C D

A. Unfractionated bulk soil

B. Control

C. Added with H2O2

D. Treated with Catalyst and H2O2

Clay

Silt

Fine Sand

Coarse Sand

OC (g.kg-1)=(gOC/kg fraction) x (kg fraction/kg bulk soil)

Emission of CO2 from control soil and soil treated with Catalyst + H2O2 ,

before and after fumigation with chloroform

(bars in graph represent standard error; n = 6)

Reduction in

emitted CO2

after fumigation

is ~50% in

respect to control

PORRARA COLOMBAIA ITRI

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2 5 days incubation

MW

Dw

(m

m)

control

polymerized


0.0

0.2

0.4

0.6

0.8

1.0

1.215 dry/wet cycles

MW

Dw

(m

m)

control

polymerized


0.0

0.2

0.4

0.6

0.8

1.0

30 dry/wet cycles

MW

Dw

(m

m)

control

polymerized

Increased structural

stability (MWDW) of three

Mediterranean soils

following an in situ

photosensitivised and

catalysed polymerization of

SOC at different wet and

dry cycles


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4C

O2 (

mg

×g

-1 o

f so

il) control

polymerized

Respiration of CO2 from control and treated soils (ton/ha)*

* mg/g = 2 ton/ha (2.000.000 Kg/Ha)

% variationVariationPolymerizedControl

-15.9-29.4-0.2-0.201.060.481.260.68ITRI

+2.0-7.4+0.06-0.162.982.002.922.16COLOMBAIA

-7.0-12.8-0.18-0.342.382.322.562.66PORRARA

30 w/d

cycles

5 days30 w/d

cycles

5 days30 w/d

cycles

5 days30 w/d

cycles

5 daysSOILS

Respiration after 5 days

incubationCO2 emissions from

three Mediterranean

Soils followin in situ

photosensivitized

polymerization of OC

catalyzed by iron-

porphyrins (10 kg.ha-1)

Considering the estimated surface of world arable land (16 x

108 ha), one application on soil of a water-soluble

biomimetic catalyzer (only 10 kg.ha) would result in an in

situ photosensitived polymerization of OC and would

reduce the CO2 emission to the atmosphere by:

3.2 x 1014 - 5.4 x 1015 gC.y-1 after 5 days

2.6 x 1014 - 3.2 x 1014 gC.y-1 after 30 w/d cycles (about

one year)

This may be a respectable amount of global CO2 reduction of

soil emission when compared to the estimated global CO2

respiration from arable soils:

8 x 1014 gC.y-1

NOT BAD!!!

CONCLUSIONS

1. HUMEOMIC or molecular mapping of HS may help in

establishing relationships between humic structure and

their biological activities on plants and help to increase

crop yields

2. Increasing the content of chemical energy in soil OM by

photo-induced polymerization under biomimetic catalysis

appears to be a very promising technological approach to

sequester carbon in soils.

There is an urgent need for soil and agricultural chemists to

devise innovative catalytic green technologies to increase

bioresistance of humic molecules in soil and reduce

greenhouse gases emission to the atmosphere.

Contribution to the research work

Riccardo Spaccini (PhD, 1999)

Pellegrino Conte (PhD, 1999)

Annunziata Cozzolino (PhD, 2003)

Anna Agretto (PhD, 2004)

Gabriella Fiorentino (PhD, 2005)

Salvatore Baiano (PhD, 2006)

Daniela Smejkalova (PhD, 2006)

Present Doctoral students :

Antonio Nebbioso, Donato Sannino, Barbara Fontaine

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