preferential accumulation of selectively preserved biomacromolecules in the humus fractions from a...

$: Preferential accumulation of selectively preserved biomacromolecules in the humus fractions from a peat deposit as seen by analytical pyrolysis and spectroscopic techniques$
Preferential accumulation of selectively preservedbiomacromolecules in the humus fractions from apeat deposit as seen by analytical pyrolysis and

spectroscopic techniques

Jose A. Gonzalez a,*, Francisco J. Gonzalez-Vila a,Gonzalo Almendros b, M. Cristina Zancada b, Oliva Polvillo a,

Francisco Martın a

a Instituto de Recursos Naturales y Agrobiologia de Sevilla, CSIC, Reina Mercedes 10, 41012 Sevilla, Spainb Centro de Ciencias Medioambientales, CSIC, Serrano, 115B, 28006 Madrid, Spain

Accepted 31 March 2003

Abstract

A series of structural features of the three main humic fractions (humic acid, fulvic acid and

humin) from different depths of a peat bog deposit in Mazagon (Huelva, Southern Spain) were

isolated and analysed by flash pyrolysis�/gas chromatography�/mass spectrometry, solid-state13C-nuclear magnetic resonance and Fourier transformed infrared spectroscopy. Such

techniques demonstrate that the various humic fractions were very different not only in terms

of molecular weight but also in their composition and structural characteristics, showing well

differentiated patterns in the relative distribution of alkyl, O-alkyl and aromatic moieties in

each humic fraction. To a large extent, selectively preserved lignin accumulates in the humic

acid (HA) fraction, whereas the fulvic acid (FA) consists of a colloidal carbohydrate with a

substantial peptidic moiety and the humin includes the noteworthy concentration of insoluble,

macromolecular polyalkyl structures. A diagnostic lignin signature in the resolution-enhanced

infrared spectra of the HA points to processes of selective preservation of macromolecular

substances derived from vascular plants. In general, the humic fractions were not extensive

sources of chemotaxonomic descriptors of previously documented biodiversity changes along

the period of the peat deposit formation. This is in harmony with a substantial homogenising

effect of diagenetic transformations throughout the whole sedimentary record with peat

* Corresponding author. Fax: �/34-95-462-4002.

E-mail address: [email protected] (J.A. Gonzalez).

J. Anal. Appl. Pyrolysis 68�/69 (2003) 287�/298

www.elsevier.com/locate/jaap

0165-2370/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0165-2370(03)00069-X

mailto:[email protected]

stratigraphy recording mostly the authigenic processes of bog formation and development.

Anthropogenic perturbations may be also responsible for the loss of information in the humic

proxy resulting in the lack of an apparent relationship between any particular input and the

composition of the humic fractions from the peat bog.

# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Histosols; Peat; Humic acids; Fulvic acids; Humin; Pyrolysis; Solid-state NMR; FT-IR

1. Introduction

Peat bog deposits were formed in peculiar paleoecosystems where the slow

biodegradation of plant residues depends on a series of pedo-climatic and

hydromorphic factors leading to a progressive accumulation of organic matter

(OM) stabilised in different evolutionary stages. Peat formation processes are slow

and the macromolecular organic components are particularly recalcitrant against

biodegradation ([1], and references therein).

From a pedological point of view, peat bog deposits are included into the

Histosols order, characterised by reduced biological activity and a complex abiotic

control of the OM transformation. The efficiency of degradation in this anoxic

environment is low, then polysaccharides are utilised preferentially over lignin [2�/4].

Due to the high performance of selective preservation processes in peat environ-

ments, the molecular composition of peat deposits may represent not only a valid

record of paleoclimatic information (usually encompassing the entire Holocene

period) but also a useful tool in paleoclimatic reconstructions. Thus, in contradiction

to the widespread view that peat stratigraphy reflects mostly the authigenic processes

of bog development, the current ideas in the field also stress the contribution of

external climatic/anthropogenic factors ([5] and references therein).

Different ways to take advantage from this geochemical information require the

study of peat stratigraphy [6], as well as the microfossil records [5�/8], which in some

cases are not present, or are not preserved at all. An alternative method is the

molecular characterisation of the different forms of OM present in the peat. In

particular, the structural characteristics of the macromolecular humic fractions have

been proved to be useful for the elucidation of diagenetic pathways during the

coalification process [9] and for obtaining information about the source of organic

materials and depositional environments [10,11]. On the other side, due to the

miscellaneous origin and variable composition of the humic materials, modern

biogeochemical research has also taken advantage of their specific molecular features

to obtain environmental information on the organisms and processes involved in soil

C sequestration mechanisms [12�/14].

Assuming the above statements, we have analysed the composition and diagenetic

processes of the OM from Laguna de las Madres peat-filled deposit (Huelva,

Southern Spain), close to the estuaries of Tinto and Odiel rivers. This ombrotrophic

bog is the most meridional among peat formations in the Northern Hemisphere, this,

together with a remarkably peculiar geomorphology [15], define a high-interest

J.A. Gonzalez et al. / J. Anal. Appl. Pyrolysis 68�/69 (2003) 287�/298288

scenario for Mediterranean paleoclimatic research. The analytical approach included

the isolation of humic acids (HA), fulvic acids (FA) and humin fractions from a peat

section taken at different depths, and their characterisation by using both solid-state13C-NMR and FT-IR spectroscopies and flash pyrolysis (Py), in combination with

gas chromatography�/mass spectrometiy (GC�/MS).

Analytical pyrolysis has been previously employed in the research on peat origin

and degree of maturity. The volatile products released during pyrolysis oftencorrespond to well known components from plant and microbial origin [16,17]. In

particular pyrolysis is an analytical tool especially responsive to the presence and

composition of lignins. In fact, the monomer composition of lignins varies greatly

within the different botanical taxa, but it also varies in terms of their degree of

diagenetic alteration in the sedimentary environment [18,19].

On the other hand, non-destructive spectroscopic techniques provide reliable

information on the relative contribution of the different structural units to the OM

fractions of the peat considered as a whole. These analytical approaches have beenpreviously used in the study of a large variety of insoluble macromolecular

substrates, including humic and lignocellulosic materials [20�/22].

In this study pyrolytic and spectroscopic analyses of peat humic fractions were

used to study the origin and diagenetic degree of the peat OM. Data obtained here

complement previous sedimentological, palynological and lipid biomarker proxies

[23�/26], revealing vegetation history, OM diagnoses and possible anthropogenic

interferences in this depositional environment.

2. Materials and methods

2.1. Sampling site

Laguna de las Madres bog is a thick peat formation of up to 5 m depth and

approximately 30 ha representing part of Mid- and the entire Late Holocene, namely

approximately 4000 years of peat accumulation. A detailed description of the peatdeposit, including vegetation and agronomical and hydrophysical properties has

been described elsewhere ([27] and references therein). Samples for this study were

collected from a 1-m depth core located in a well preserved area at the margin of the

lagoon. Two sections at 30�/40 and 70 cm were taken and dried at room temperature.

2.2. Isolation of organic matter fractions

The peat OM was separated into lipid, alkali-soluble and particulate (humin)fractions following standard procedures in humus chemistry [27,28]. Total organic

carbon and organic carbon distribution into the different fractions were determined

by wet oxidation with potassium dichromate.

The isolation of the extractable humic fractions was carried out after previous

extraction of lipid with 40�/60 8C petroleum ether. The peat residue was then

successively treated with water, to isolate the water-soluble fraction, and then with

J.A. Gonzalez et al. / J. Anal. Appl. Pyrolysis 68�/69 (2003) 287�/298 289

0.1 M Na4P2O7 and 0.1 M NaOH, five-times each. The dark brown suprnatant

solution (total humic extract) was then precipitated by dropwise addition of 6 M

HCl to separate the acid-soluble FA from the acid-insoluble HA. The latter fraction

was then redissolved in 0.5 M NaOH and centrifuged at 43 500�/g , reprecipitated,

dialysed in cellophane bags, and desiccated at 40 8C.

The insoluble peat fraction (humin) was purified by 1 M HCl�/HF treatments at

room temperature, then dialysed for 1 week and desiccated.

2.3. Pyrolysis�/gas chromatography�/mass spectrometry

The FA, HA and humin fractions were analysed by Curie-point pyrolysis using a

Horizon Instruments device attached to a Varian Saturn 2000 GC/MS system.Samples were heated on ferromagnetic wires at 510 8C for 5 s. The interface of the Py

unit was set to 250 8C, and the gas chromatograph was programmed from 50 to

100 8C at 32 8C min�1 and then up to 320 8C at a rate of 6 8C min�1. The injection

port, attached to a liquid CO2 cryogenic unit, was adjusted from �/308 C (1 min) to

300 8C at 20 8C min�1. A fused-silica capillary column (25 m�/0.32 mm�/0.4 mm)

coated with CPSil was used. Mass spectra were acquired with a 70 eV ionising

energy. The identification of individual compounds was achieved by single ion

monitoring for different homologous series, low-resolution mass spectrometry andcomparison with published and stored (NIST and Wiley libraries) data.

2.4. Solid-state 13C-NMR spectroscopy

Solid-state 13C-NMR analyses of the fractions were performed on a Bruker DSX200 spectrometer (Bruker Analytische Messtechnik, Rheinstetten, Germany) oper-

ating at a 13C resonance frequency of 50.3 MHz. A commercial Bruker double-

bearing probe with 7-mm outer diameter and phase-stabilised zirconium dioxide

rotors was used. The cross-polarisation (CP) technique was applied during magic-

angle spinning (MAS) of the rotor at 6.8 kHz. A contact time of 1 ms was used. At

about 50 000 scans were accumulated using a pulse delay of 250 ms. Prior to Fourier-

transformation, a line broadening between 30 and 100 Hz was applied to determine

the relative C distribution. The spectra were integrated across four major chemicalshift regions, assignable to alkyl C (0�/45 ppm), N- and O-alkyl C (45�/110 ppm),

aromatic C (110�/160 ppm) and carboxyl/carbonyl C (160�/220 ppm).

2.5. Fourier-transformed IR spectroscopy

The Fourier-transformed infrared (FTIR) spectra were acquired with a Bruker

IFS28 spectrophotometer using KBr pellets with 2 mg sample. The spectra in digital

format were subjected to a procedure for resolution enhancement based on the

subtraction of the raw spectrum from a positive multiple of its second derivative

[29,30].


Table 1

General analytical characteristics and distribution of total OM in different fractions isolated from Laguna de las Madres peat at 30�/40 and 70 cm

Sample

depth

Typea Organic C

(%)

N

(%)

C/

N

Lipidb Water-soluble

OMb

Total humic ex-

tractb

Humic acid

(HA)b

Fulvic acid

(FA)b

Huminb HA/

FA

30�/40 cm Fibric 38.0 (100) 1.87 24.3 3.08 0.75 (1.97) 15.32 (40.3) 10.87 (28.6) 4.45 (11.7) 21.92

(57.69)

2.44

70 cm Hemic 34.5 (100) 1.81 26.2 2.99 0.46 (1.33) 13.66 (39.6) 9.03 (26.2) 4.63 (13.4) 20.37

(59.05)

1.95

In brackets, percentage of the total organic carbon.a According to the size fractionation and the respective content in fibres.b g/100 g sample.

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3. Results and discussion

Table 1 shows the total OM content and its quantitative distribution into the

different fractions in the two peat sections studied. A slight decrease with depth of

both total OM and humic fractions is observed. The relatively high concentrations of

lipids and the presence of water-soluble material reflect a low decomposition degree.

This is also in agreement with the low content in humic fractions and low

humification quality [27].Fig. 1 and Table 2 shows the solid-state 13C-NMR spectra obtained from the three

humic fractions and the quantitative distribution of the different C-types. The

spectra showed typical signals centred at 172 ppm (carbonyl carbons), 56 ppm

(methoxyl and a-amino carbons) most prominent signals correspond to (172 ppm),

33 ppm (signal assigned to alkyl carbons in polymethylene structures from lipid

polymers or condensed wax material) and the peaks for C1 (105 ppm) and C2, C3,

C5 (73 ppm) carbons in sugar-derived structures.

The integration values of the different C-types only reflected very small differences

between the humic fractions isolated downcore. However, outstanding differences

were found in the distribution patterns of the different C types in each humic

fraction In particular the predominantly O-alkyl character of the structures present

in the FA fraction (similar to a COOH containing, selectively preserved carbohy-

drate) and the dominance of aliphatic, mainly alkyl, structures in the humin were

Fig. 1. Solid-state 13C-NMR spectra of the humic fractions extracted from Laguna de las Madres peat.


evident. The presence of N-containing structures cannot be straightforwardly

recognised in the 13C-NMR because signals corresponding to amide structures

overlap those produced by alkyl, carbonyl and methoxyl structures (i.e. 33, 172 and

56 ppm).

The infrared spectra (Fig. 2) of the HA fraction show a featureless profile

corresponding to a heavily transformed macromolecular mixture. Nevertheless, after

Table 2

Solid-state 13C-NMR of the humic fractions extracted from Laguna de las Madres peat, quantitative

distribution of the different C-types

Integrated region (ppm) Fulvic acid Humic acid Humin

220�/161 Carbonyl/carbonyl C 13.729/0.24a 14.569/1.97 8.269/0.38

161�/141 Hydroxy aromatic C 4.059/2.01 8.419/0.60 5.539/0.38

141�/110 Aromatic C 9.119/3.38 20.129/2.85 13.979/1.16

110�/45 O/N-alkyl C 52.949/5.07 23.449/2.16 35.879/5.32

45�/(�/10) Alkyl C 20.169/4.87 33.449/7.51 36.339/5.85

a Standard deviation (n�/3).

Fig. 2. Detail of the 2000�/800 cm�1 region of the resolution-enhanced infrared spectra of humic and FAs

(the original spectra are superimposed).


resolution enhancement the spectra showed a fairly well-defined band pattern with

diagnostic peaks centred at 1510, 1460, 1420, 1270 and 1230 cm�1, which is

interpreted as the presence of a substantial domain of selectively preserved lignin

[31,32].

No substantial differences downcore were found when analysing FT-IR spectra of

the isolated humic and FA fractions. Nevertheless, this pattern tends to be ill-defined

in the underlying 70 cm sample, as it could correspond to a more intense

transformation of plant-derived macromolecules. In addition, the spectrum shows

a low intensity alkyl stretching band (2920 cm�1) and a well-defined 1720 cm�1

carboxyl band. On the other hand, the FA spectra show no straightforwardly

recognisable lignin pattern but intense peaks centred at 1070 cm�1, suggesting a

substantial carbohydrate moiety. The resolution-enhanced spectra show a broad

band with maximum at approximately 1620 cm�1, which has probably a

miscellaneous origin [33]. Apart from the likely contribution of some aromatic

and quinoid structures to this band, the presence of ill-defined shoulders near 1550

and 1660 cm�1, to some extent recognisable in the resolution-enhanced spectrum

points to the contribution of amides coming from preserved proteinaceous materials

in the FA fraction.

Fig. 3 shows the Curie-point pyrolytic profiles of the various humic fractions

isolated from the peat core at 70 cm depth. The chemical identity and source

assignation of the major pyrolysis products is reported in Table 3. Upon pyrolysis,

the FA released typical anhydrosugar and furan compounds, as well as a wide set of

Fig. 3. Curie-point pyrograms of the HA, FA and humin fractions extracted from Laguna de las Madres

peat. The numbers on the peaks refer to Table 3; C-range refers to fatty acids.


N-containing products, respectively, arising from carbohydrate-derived and peptidic

structures.The HA fraction released typical methoxyphenols with both guaiacyl and syringyl

skeletons, pointing to the presence of a microbially reworked, not-extensively

demethoxylated lignin. The pyrolytic methoxyphenols have chemotaxonomic inter-

Table 3

Chemical identity of the pyrolysis products (other than long chain alkyl compounds) released from the HA

and FA fractions

Fulvic acids Humic acids

1 Pyrrole [Pr] 24 Pentanedione [Ps] 49 1-(4-Hydroxyphenyl) etha-

nol [Lig]

2 Toluene [Lig/Pp] 25 C2-Alkylbenzene [Lig/Pp] 50 Trans -4-propenylphenol

[Lig]

3 Furanone [Ps] 262-Methyl-2-cyclopenten-1-one

[Ps]

51 5-Ethylpyrogallol [Lig]

4 Imidazole [Pr] 27 2-Acetylfuran [Ps] 52 4-Propylguaiacol [Lig]

5 Furaldehyde [Ps] 28 Isomer of 22 [Ps] 53 3-Methylindole [Lig]

6 Methylpyrrole [Pr] 29 5,6-Dihydropyran-2,5-dione

[Ps]

54 Vanillin [Lig/Pp]

7 Methylpyridine [Pr] 30 4 Hydroxy-5,6-dihydro-(2H)-

pyran-2-one [Ps]

55 Cis -isoeugenol [Lig]

8 Methylcyclopentenone [Ps] 31 2-Hydroxy-3 methyl-2 cyclo-

penten-1-one [Ps]

56 4-Hydroxyacetophenone

[Lig]

9 Styrene [Pp, Lig] 32 Methylphenol [Lig/Pp/Pr] 57 2,6-Dimethoxy-4-methyl-

phenol [Lig/Pp]

10 Dimethylpyrrole [Pr] 33 Isomer of 32 [Lig/Pp/Pr] 58 Trans -isoeugenol [Lig]

11 Dimethylpyridine [Pr] 34 Guaiacol [Lig] 59 Acetovanillone [Lig]

12 Methylfurancarboxaldehyde [Ps] 35 Phenylacetonitrile [?] 60 Vanillic acid methyl ester

[Lig]

13 Phenol [Lig/Pp/Pr] 36 2,4-Dimethylphenol [Lig/Pp] 61 4-Ethyl-2,6-dimethoxyphe-

nol [Lig/Pp]

14 Cyanobenzene [Pr] 37 Benzoic acid [Lig/Pp] 62 Guaiacylacetone [Lig]

15 Hydroxymethylcyclopentenone

[Ps]

38 4-Ethylphenol [Lig/Pp] 63 Vanillic acid [Lig]

16 Hydroxydihydropyranone [Ps] 39 3,4-Dihydroxybenzaldehyde

[Lig/Pp]

64 Propiovanillone [Lig]

17 Pyrrole-2-carboxaldehyde [Pr] 40 Catechol [Lig/Pp] 65 2,6-Dimethoxy-4-propyl-

phenol [Lig/Pp]

18 2-Hydroxy-3-methyl-2-cyclopen-

ten-1-one [Ps]

41 4-Vinylphenol [Lig/Pp] 66 Acetosyringone [Lig]

19 Dimethylhydantoin [?] 42 4-Allylphenol [Lig/Pp] 67 Isomer of 66 [Lig]

20 4-Ethylphenol [Lig/Pp] 43 3-Methoxycatechol [Lig/Pp] 68 Syringylacetone [Lig]

21 Levoglucosenone [Ps] 44 4-Ethylguaiacol [Lig] 69 Syringic acid [Lig]

22 Hydroxymethylfuraldehyde [Ps] 45 4-Methylcatecbol [Lig/Pp] 70 Propiosyringone [Lig]

23 Hydroxypyridine [Pr] 46 Indole [Pr] 71 Syriyngyl vinyl ketone [Lig]

47 Cis -4-propenylphenol [Lig] 72 Ferulic acid [Lig]

48 4-Vinylguaiacol [Lig]

Probable source of the different Py products is indicated in brackets: Pr, protein; Lig, lignin; Ps,

polysaccharide; Pp, polyphenols ([34�/39] and references therein).


est and have been previously used as sedimentary source indicators [40], since their

relative concentrations provide information on the extent to which plant-inherited

macromolecules are selectively preserved or substituted by microbially reworked or

newly formed substances.

The humin fractions yielded a high variety of alkyl molecules in agreement with

previous degradative studies, suggesting the accumulation in this fraction of

recalcitrant, insoluble, lipid polymer macromolecular material [41].In general, a correlation between pyrolytic (GC�/MS�/Pyr) and spectroscopic (13C-

NMR and FT-IR) data was found, and thus the reliable quantitative distribution of

C atoms pertaining to alkyl and aromatic structures obtained from NMR was in

acceptable agreement with the relative quantification of the pyrolysis products.

4. Conclusions

The results obtained support the view that different humic fractions originate from

relatively different precursors and were probably affected by independent mechan-

isms of selective preservation, diagenetic alteration, and microbially reworking,

being then incorporated in a different manner to the humic fractions. In particular,the humin fraction is enriched in insoluble alkyl material, revealed by both NMR

and mainly Py�/GC�/MS, whereas the HA fraction is enriched in altered lignin and

there is evidence for the preferential incorporation of carbohydrate, possibly low-

molecular weight polysaccharides (hemicelluloses), into the FA fraction, which also

contains some additional amount of N-containing compounds.

No qualitative fluctuations downcore were observed in the spectral and the

pyrolytic patterns of the different humic fractions. Consequently no clear changes in

the molecular assemblages of signature compounds along the period of the peatdeposit formation could be unambignously shown, which could in turn be used to

determine possible climatic or environmental changes.

In accordance with the view that peat stratigraphy records should reflect the

authigenic processes of bog development, it is probable that the peat formation from

Laguna de las Madres under study behaves as a highly resilient soil subsystem for the

whole peat formation period (Late-Holocene). It is a strong ombrothrophyc system,

where specialised hydrophytic vegetation and cycling hydromorphism, mostly

dependent on the microtopographic features of the area, have probably determineda peculiar internal/endogenous? dynamics, to a large extent irresponsive to the

general climatic and phytosociological changes that took place in the neighbouring

ecosystems.

Acknowledgements

This research has been supported by the Spanish CICyT under grants AMB99-

0907 and AMB99-0226-02. J.A. Gonzalez acknowledges financial support from the


Spanish Ministry of Science and Technology and the European Regional Develop-

ment Fund (‘‘Ramon y Cajal’’ program).

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