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Organic Geochemistry 38 (2007) 2012–2023

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OrganicGeochemistry

The complementary use of 1H NMR, 13C NMR, FTIRand size exclusion chromatography to investigatethe principal structural changes associated with

composting of organic materials with diverse origin

Marta Fuentes a, Roberto Baigorri b, Gustavo Gonzalez-Gaitano a,Jose Ma Garcıa-Mina a,b,*

a Department of Chemistry and Soil Chemistry, University of Navarra, 31080 Pamplona, Spainb CIPAV-Roullier Group, Inabonos, Polıgono Arazuri-Orcoyen, 31160 Orcoyen, Spain

Received 5 December 2006; received in revised form 27 July 2007; accepted 23 August 2007Available online 31 August 2007

Abstract

The aim of this work is to study the structural changes involved in humification processes. Total humic extracts (THE)obtained from five composted materials of diverse origin (solid wastes of wineries, solid mill olive wastes, domestic wastes,ovine manures plus straw, and a mixture of animal manures), and their corresponding initial raw fresh organic mixtureswere studied using 13C nuclear magnetic resonance (NMR) using the cross-polarization magic angle spinning technique(CPMAS), 1H NMR, Fourier transform infrared spectroscopy (FTIR) and high pressure size exclusion chromatography(HPSEC). One group of three humic acids extracted from soils, and a second group consisting of two reference humic acidsand two reference fulvic acids (1S104H, 1R103H, 1R101F and 1R107F) obtained from the International Humic Sub-stances Society were also characterized using these techniques, in order to compare the features of reference humic andfulvic acids with those of composted materials. Likewise, the results were compared with those obtained in previous stud-ies, in which UV–Visible and fluorescence spectroscopies were employed to characterize the humification degree of themolecular systems.

The results obtained by 13C CPMAS NMR, 1H NMR and FTIR indicate that, in general, humification seems to beassociated with an increase in the aromatic character of the systems, with the presence of phenol groups as principal sub-stituents and a reduction in oxygen containing functional groups, principally carboxylic or carbonylic groups, as well asthe development of molecular fractions with larger size. These results also support the suitability of UV–Visible and fluo-rescence spectroscopies in the assessment of the humification course of humic extracts in composting processes.� 2007 Elsevier Ltd. All rights reserved.

0146-6380/$ - see front matter � 2007 Elsevier Ltd. All rights reserveddoi:10.1016/j.orggeochem.2007.08.007

* Corresponding author. Address: Department of Chemistryand Soil Chemistry, University of Navarra, 31080 Pamplona,Spain. Tel.: +34 948324550; fax: +34 948324032.

E-mail address: jgmina@inabonos.com (J.M. Garcıa-Mina).

1. Introduction

Recycling organic wastes from industrial anddomestic sources by means of composting has arisenin recent years as an alternative to classical soil

.

M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023 2013

amendment (Gonzalez-Vila et al., 1999; Ait Baddiet al., 2004a,b; Amir et al., 2004). These organic res-idues have to be pretreated in order to stabilize themand to reduce their initial toxicity. Non-compostedresidues or immature composts applied to agricul-tural soils degrade rapidly, and may cause phytotox-icity due to the presence of simple organic acids, anddecreases in oxygen and nitrogen concentrations,among other harmful effects on soil properties(Mustin, 1987; Jimenez and Garcıa, 1992). The con-tent in potential organic (i.e., polycyclic aromatichydrocarbons) and inorganic (heavy metals) pollu-tants of composts must also be controlled, as thesecontaminants may migrate to groundwater or accu-mulate in plants (Oleszczuk and Baran, 2005; Alvar-enga et al., 2007; Oleszczuk, in press).

The quality and stability of composts depend lar-gely on the properties of the initial materials, thetime of composting and the oxidative conditionsof treatment that influence microbial activities,resulting in different degrees of degradation andtransformation of the initial organic mixtures. Com-posting has been described as an accelerated versionof the decomposition processes naturally occurringin the soil (Ait Baddi et al., 2004a). Therefore, thecharacterization of the changes in the compositionof the compost and its comparison with referencehumic substances generated in soils are two impor-tant points to consider when assessing the qualityand properties of the final products, and studyingthe singular structural changes and features associ-ated with humification. These changes have classi-cally been attributed to increases in aromaticcontent in humic substances, although other studieshave shown that accumulation of recalcitrant ali-phatic structures also takes place during composting(Almendros et al., 2000).

Organic matter transformation during com-posting can be estimated by C/N ratios, solubleorganic carbon concentration in water extracts orhumic acid to fulvic acid ratio (Hsu and Lo, 1999;Jouraiphy et al., 2005). Indices derived from fluores-cence and UV–Visible spectroscopies have also pro-ven to be useful in evaluating the humificationdegree of HS extracted from organic materials ofdiverse origin (Korshin et al., 1997; Peuravuori andPihlaja, 2002; Milori et al., 2002; Fuentes et al.,2006), as well as Fourier transform infrared spectros-copy (FTIR) and nuclear magnetic resonance spec-troscopies (NMR) (Gonzalez-Vila et al., 1999;Sanchez-Monedero et al., 2002; Ait Baddi et al.,2004a; Amir et al., 2004; Jouraiphy et al., 2005).

In a previous study (Fuentes et al., 2006), extractsobtained with alkaline solutions from several com-posted materials and from their initial raw organicmixtures, in addition to various humic and fulvicacids from soils, including International HumicSubstances Society (IHSS) reference materials, werestudied using UV–Visible and fluorescence spectros-copies. Several indices indicated reasonably well theevolution (humification) of the different systems,indicating that humification might be related to:(i) significant changes in the aromatic character ofthe samples, which in turn could be associated withmore functional ring substitution and polyconden-sation; and (ii) significant changes in the molecularsize distribution throughout humification.

In order to study more in depth these possiblestructural changes associated with humification,we have investigated the same organic systems char-acterized by UV–Visible and fluorescence spectros-copies (Fuentes et al., 2006) through thecomplementary use of elemental analysis, 1HNMR, 13C NMR, FTIR and high pressure sizeexclusion chromatography (HPSEC). Likewise, thisstudy has permitted us to compare the properties ofcomposted materials with those of humic and fulvicacids extracted from soils.

2. Materials and methods

2.1. Organic materials

The organic systems studied were divided intothree groups: (i) humic substances (humic and fulvicacids) with diverse origin, including IHSS stan-dards; (ii) organic substances contained in alkalineextracts obtained from composted materials; and(iii) organic substances contained in alkalineextracts obtained from the initial organic materialsused in the different composting procedures.

Soil humic substances were isolated from soilsamples of different locations: China (CHHA) andCzech Republic (CZHA). Commercial humic acidwas obtained from Aldrich Chemicals (AHA),whereas Leonardite Standard Humic Acid (LSHA),Pahokee Peat Reference Humic Acid (PRHA),Suwannee River Reference Fulvic Acid (SRFA)and Waskish Peat Reference Fulvic Acid (WRFA)were purchased from IHSS (codes: 1S104H,1R103H, 1R101F and 1R107F, respectively).

Total humic extracts (THE) obtained from com-posted and non-composted raw materials weredefined in pairs: compost of solid wastes of wineries

2014 M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023

(GWC) and initial solid wastes of wineries (GW);compost of solid mill olive wastes (OLVC) and ini-tial mill olive residues (OLV); compost of domesticwastes (DWC) and initial domestic wastes (DW);compost of ovine manures plus straw (OVC) andstraw (STW); compost of a mixture of animalmanures (FMC) and the initial mixture (non-composted) of animal manures (FM). The charac-terization of these materials is summarized in Table1. The starting materials were piled in rows, andthese piles were periodically turned over in orderto aerate and homogenize the mixtures. The timeof composting was four months, and during thisperiod piles were turned six times.

2.2. Extraction procedure

The different organic systems were extracted fromsolid samples of the organic materials with 0.1 MNaOH (24 h of mechanical shaking in darkness) at22 �C. The air was displaced by N2 in the extracts inorder to avoid possible oxidation during the extrac-tion process. The sample:extractant ratio used was1:6 for all samples but 1:10 for straw (this needed alarger volume of NaOH solution because of its greatliquid absorption capacity). The suspension was thencentrifuged at 11,100 g for 15 min and the alkalinesupernatants were treated with an acidic-cationexchange resin (Amberlite IR-120H, Aldrich) inorder to lower the pH to 3.5 before freeze-drying.

2.3. Elemental analysis

The carbon, hydrogen and nitrogen contents ofthe lyophilized samples were analysed in duplicate

Table 1Moisture, ash content and elemental composition (w/w %) of the initia

Organic substances Moisturea Ashb

Ovine manure STW 8.9 0.6OVC 8.0 7.0

Animal manure FM 8.0 11.1FMC 4.6 10.7

Olive wastes OLV 9.1 2.5OLVC 8.4 4.0

Grape wastes GW 9.5 3.1GWC 12.0 5.5

Domestic wastes DW 7.2 2.7DWC 8.4 7.4

a Wet matter.b Dry matter.c Dry and ash-free matter.

by a LECO CHN 900 analyser. The oxygen contentwas determined by difference (ash-free basis).

2.4. 13C NMR spectroscopy

13C NMR spectra were obtained on a VarianUnity 300 spectrometer at 75.429 MHz using thecross-polarization magic angle spinning technique(CPMAS), with a spinning speed of 5 kHz, 90� pulsewidth, 69 ms acquisition time and 1.0 s delay.

2.5. 1H NMR spectroscopy

Solution 1H NMR spectra were recorded on aVarian Unity-300 spectrometer at 300 MHz, usinga 5 mm multinuclear probe, with 90� pulse angle,a sweep width of 4000 Hz and a line broadeningof 0.5 Hz. Gated irradiation was applied betweenacquisitions to presaturate the residual water peak.Sodium 3-trimethylsilyl-propionate-2,2,3,3,-d4 (TSP)was added to the samples to provide a chemical shiftstandard.

2.6. FTIR spectroscopy

Pellets were prepared by mixing 1 mg of eachfreeze-dried sample with 100 mg of KBr so that themixture became homogeneous. Infrared spectra wererecorded on these pellets with a Nicolet Magna-IR550 spectrometer over the 4000–400 cm�1 range.

2.7. HPSEC study

Molecular size distribution for humic materialswas evaluated by high performance size exclusion

l organic mixtures and the corresponding composted materials

Cc Hc Nc Oc C/N C/H

48.2 5.8 0.3 45.6 177.9 0.6951.3 5.3 4.3 39.1 14.0 0.81

57.4 6.2 8.0 28.4 8.4 0.7753.3 6.2 7.2 33.4 8.7 0.72

54.0 6.5 1.5 38.0 41.3 0.6956.2 5.2 2.6 36.0 25.5 0.90

52.4 5.7 1.9 40.0 32.7 0.7758.0 5.1 3.7 33.2 18.3 0.94

43.2 4.6 1.3 50.9 38.7 0.7854.5 5.6 4.8 35.1 13.3 0.82

M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023 2015

chromatography. The chromatographic system con-sisted of a Waters 600 Controller pump followed bytwo detectors in series: a Waters 996 PhotodiodeArray Detector set at 400 nm, and a Waters 2424refractive index detector (RI). Size exclusion separa-tion occurred through a PL Aquagel-OH 30 column(Polymer Laboratories), preceded by a guard col-umn with the same stationary phase. The overallmolecular weight range of separation for this col-umn is 100–300,000 Da.

For each sample, solutions of 800 mg of organiccarbon l�1 were prepared in 0.05 M NaNO3. Theinjection volume of all samples was 100 ll, the elu-ent used was 0.05 M NaNO3 (pH 7), and the flowrate was 1 ml/min. Void volume (V0 = 6.65 ml)and permeation volume (Vp = 11.82 ml) were deter-mined with polyethylene oxide of MW of 43,250 Daand methanol, respectively.

3. Results and discussion

The results obtained using the different tech-niques will be presented and commented upon sep-arately, and afterwards these results will be relatedto those previously obtained using UV–Vis andfluorescence spectroscopies as described in Fuenteset al. (2006).

Table 2Elemental analysis for HAs and FAs from soils and IHSS and for humcomposts (see acronyms in the text, Section 2.1)

Families Organic substances Elemental analysis

% C % H

HS CHHA 59.6 1.6CZHA 59.4 2.4AHA 53.4 3.7

HS-IHSS LSHA 62.2 3.6PRHA 55.7 3.8SRFA 52.5 4.3WRFA 53.5 4.2

Ovine manure STW 40.1 3.7OVC 33.7 3.0

Animal manure FM 43.2 4.4FMC 34.2 3.7

Olive wastes OLV 56.0 5.0OLVC 48.7 4.1

Grape wastes GW 38.9 3.9GWC 48.1 3.2

Domestic wastes DW 39.1 5.1DWC 41.4 3.3

3.1. Elemental analysis

The HA studied (CHHA, CZHA, AHA,LSHA, PRHA) presented the highest content incarbon, around 55–60% (Table 2). Reference ful-vic acids showed slightly lower values, but theyhad higher oxygen content, mainly due to the lar-ger content in COOH groups in those compoundsas shown by 13C NMR (Table 3). Values for O/Catomic ratios fell within the range described in theliterature for HA and FA. Thus, HAs presentedO/C values around 0.5, whereas those of FAswere close to 0.7 (Stevenson, 1994). Differencesin the nitrogen content were not significant,although C/N ratio varied, being lower in HAsand indicating larger nitrogen fixation in thesesubstances.

As for the organic substances extracted fromcomposted and non-composted materials, the car-bon content decreased in OVC, OLVC and FMC,but increased in GWC and DWC. The percentageof H and C/N ratios decreased in all cases. TheC/H ratios increased in all cases to varying degrees,while the N content increased in all cases exceptFMC.

The increase in the C/H ratio suggests that chan-ges in chemical composition during the composting

ic-like substances extracted from raw initial organic mixtures and

Atomic ratios

% N % O O/C C/N C/H

1.4 37.4 0.47 49.7 3.101.3 36.8 0.46 53.3 2.060.7 42.2 0.59 89.0 1.20

1.2 30.5 0.37 60.5 1.443.6 36.9 0.50 18.0 1.220.7 43.5 0.62 87.5 1.021.1 41.7 0.58 56.7 1.06

0.6 55.5 1.04 78.0 0.903.7 59.5 1.32 10.6 0.94

10.0 42.4 0.74 5.0 0.827.7 54.5 1.19 5.2 0.78

0.6 38.4 0.51 108.9 0.932.5 44.7 0.69 22.7 0.99

1.0 56.3 1.08 45.4 0.834.6 44.1 0.68 12.2 1.25

1.4 54.4 1.04 32.6 0.637.1 48.2 0.78 6.8 1.04

Table 3Relative abundances of different carbon and hydrogen types (in %) measured by 13C CPMAS NMR and 1H NMR spectroscopy, and parameters obtained by UV–Vis and fluorescencespectroscopies

Families Organic substances 13C NMR (Chemical shift range in ppm) 1H NMR (Chemicalshift range in ppm)

UV–Vis and fluorescenceparametersa

0–45 45–110 110–160 140–160 160–215 0.5–3 3–4.5 6–8.5 HarH/CarC EET=EBzb e280

c e600d A440

e

Alkyl C O-alkyl C Aromatic C Phenolic C Carbonylic C Hal Ha Har

HS CHHA 13.8 3.2 79.5 5.4 3.5 24 53 23 0.09 0.90 980 92.5 10230CZHA 27.4 12.6 46.5 12.8 13.5 70 17 13 0.13 0.86 1064 51.9 16160AHA 62.5 18.8 12.1 5.3 6.6 42 16 42 3.03 0.84 777 50.6 8110

HS-IHSS LSHA 26.6 14.9 48.7 12.8 9.8 31 68 1 0.01 0.82 1402 80.1 8540PRHA 20.1 10.9 37.3 12.4 31.7 52 40 8 0.18 0.77 834 58.3 5970SRFA 32.5 14.2 15.6 5.0 37.7 71 22 7 0.47 0.62 449 46.5 4490WRFA 23.2 12.2 29.0 9.8 35.6 37 36 27 0.88 0.67 500 16.3 4750

Ovine manure STW 18.6 49.7 8.2 3.3 23.5 26 65 9 1.21 0.45 116 2.0 1010OVC 17.7 25.0 22.3 10.0 35 48 46 6 0.26 0.50 373 14.7 4150

Animal manure FM 33.7 13.8 11.8 4.0 40.7 72 24 4 0.45 0.41 108 3.0 2040FMC 30.4 24.0 9.6 3.0 36 27 68 5 0.68 0.56 286 9.8 4110

Olive wastes OLV 23.2 56.4 11.2 5.4 9.2 40 58 2 0.23 0.47 106 13.2 1840OLVC 22.2 29.3 19.7 7.1 28.8 45 47 8 0.42 0.65 256 16.9 4550

Grape wastes GW 3.5 46.9 1.5 1.0 48.1 28 70 2 1.33 0.33 55 3.1 780GWC 21.6 13.1 26.8 10.0 38.5 56 43 1 0.15 0.76 415 19.5 4480

Domestic wastes DW 23.7 28.2 20.3 9.5 27.8 61 36 3 0.26 0.51 70 2.8 680DWC 25.4 34.9 16.4 5.0 23.3 56 39 5 0.32 0.62 354 26.4 3830

a Values presented for EET/EBz, e280, e600 and A440 are taken from a previous study (Fuentes et al., 2006).b Ratio of absorbances at 253 and 220 nm in the UV spectrum.c Molar absorptivity at 280 nm (L cm�1 mol�1 of organic carbon).d Molar absorptivity at 600 nm (L cm�1 mol�1 of organic carbon).e Area under fluorescence emission spectra (460–650 nm) with kexc = 440 nm.

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M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023 2017

process could be due to the degradation of sub-stances with low C/H, such as carbohydrates, poly-saccharides or fatty acids, along with an increase inunsaturated structures relative to saturated ones(Miiki et al., 1997; Sanchez-Monedero et al.,2002). Nitrogen compounds are thought to be incor-porated to the humic ‘‘core’’ through condensationof proteins and modified lignin, reactions betweenN-containing compounds and quinones derivedfrom lignin, or sugar-amine condensation (Steven-son, 1994).

The absence of significant differences in thechemical composition corresponding to the organicsubstances extracted from fresh and compostedmaterials has also been found by other authors(Riffaldi et al., 1983; Inbar et al., 1990; Ait Baddiet al., 2004a), who reported that the chemical com-position of humic-like substances extracted fromvarious organic sources at different stages of matu-rity remained almost unchanged duringcomposting.

On the other hand, the organic substancesextracted from composted materials presentedC/H ratio values similar to those of fulvic acidsand lower than those of humic acids. These resultscould indicate the presence of less aromatic struc-tures in the organic substances extracted fromcomposted materials compared to the soil humicacids. Finally, the high nitrogen values found inthe organic substances extracted from compostedmaterials could be indicative of high contents ofnon-humified biomolecules (such as polysaccha-rides and polypeptides) or to the incomplete hydro-lysis of proteinaceous constituents in these systems(Sanchez-Monedero et al., 2002; Ait Baddi et al.,2004a).

3.2. 13C NMR study

In general the main trends inferred from the ele-mental analysis study were confirmed by the resultsobtained in the 13C NMR study.

The 13C NMR spectra are shown in Fig. 1. Fordata analysis, spectra were divided into chemicalshift regions assigned to the following classes ofchemical groups: alkyl C (0–45 ppm), O-alkyl C(45–110 ppm), olefinic and aromatic C (110–160 ppm), phenolic C (140–160 ppm), and carbonylC (160–220 ppm). The relative intensity of theseregions was determined by the integration of thecorresponding peak areas (Malcolm, 1989; Steven-son, 1994; Gonda et al., 2005).

Significant changes were observed betweenGWC, OLVC, OVC and their respective initial freshorganic materials GW, OLV, STW. In the threecases the aliphatic content decreased due to a lossof alkyl carbon bonded to oxygen, whereas percent-ages of aromatic, phenolic, and carbonylic Cincreased (Table 3). These changes may reflect thatduring composting, the unstable organic com-pounds such as aliphatic materials are transformedthrough intense microbial activities into more stablehumic compounds with more oxidized, olefinic oraromatic structures that could include more poly-condensed rings (Amir et al., 2004).

In the case of DW–DWC and FM–FMC sys-tems, however, the distribution of functionalgroups did not undergo important variations,which mainly consisted of an increase in aliphaticC (mainly O-alkyl C) and a slight decrease in aro-matic and carbonylic C (Table 3). Gonzalez-Vilaet al. (1999) studied composts from urban wastesand did not distinguish a clear progressive trendof the organic components in the course of thecomposting process. Composting led to simulta-neous degradation of all types of C, with no selec-tive accumulation of any preferentially stableforms. Almendros et al. (2000) found that in mostcases for composted forest and shrub biomass, therecalcitrant material accumulated during com-posting was not exclusively aromatic in natureand that the presence of tannins may contribute,through their selective preservation and condensa-tion reactions, to limiting the decomposition ofaliphatic biomacromolecules. Likewise, theseauthors indicated that the decrease in the carbox-ylic content might be due to the degradation oflipids. These authors also emphasize the possibleinterfering signals of protein moieties in the humicsubstances extracted from composts, as aminoacids contribute to resonance in the alkyl C, O-alkyl C and carbonyl C regions.

On the other hand, soil humic acids showedlower aliphatic content (mainly methyl and methy-lene groups) and higher aromatic content whencompared with the organic substances extractedfrom composted materials, except in the case ofthe Aldrich humic acid (AHA) that was mostly ali-phatic, in agreement with what had been previouslyreported (Malcolm and MacCarthy, 1986; Shinet al., 1999). It was noteworthy the similarities infunctional groups between the organic substancesextracted from composted materials and the IHSSfulvic acids.

225 200 175 150 125 100 75 50 25 0

WRFA

SRFA

PRHA

LSHA

AHA

CZHA

CHHA

δ (ppm)

225 200 175 150 125 100 75 50 25 0

STW

FMFM

OVC

FMC

OLV

OLVC

GW

GWC

DWC

DW

δ (ppm)

a b

Fig. 1. 13C CPMAS NMR spectra of: (a) HAs and FAs from soils and IHSS; and (b) humic extracts from non-composted and compostedorganic materials.

2018 M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023

3.3. 1H NMR study

Several representative 1H NMR spectra recordedfor the different systems studied are shown in Fig. 2.Proton spectra are typically divided into three mainregions: (i) 0.5–3 ppm, resonance of alkyl protonsand protons attached to carbon in a to aromatic

8 0

D2O peak

PRHA

WRFA

OLV

OLVC

δ (ppm)246

Fig. 2. 1H NMR spectra of a humic acid (PRHA), a fulvic acid(WRFA), and the humic extracts from a raw initial mixture(OLV) and its corresponding composted material (OLVC).

ring, carboxyl and carbonyl groups (Hal); (ii) 3–4.5 ppm, protons attached to carbon bearing oxy-gen or nitrogen (Ha); and (iii) 6–8.5 ppm, protonsattached to unsaturated carbons and aromatic pro-tons (Har) (Lambert and Lankes, 2002). The inte-gration of these areas and the percentage of eachtype of proton are shown in Table 3.

In the region of resonance of aliphatic protons,peaks around 0.9 ppm are assigned to protons ofterminal methyl groups, whereas signals between1.2 and 1.8 ppm are attributed to methylene andmethine protons. The peaks appearing in the rangeof 2.0–2.8 ppm can be assigned to protons adjacentto functional groups with electronegative atoms(carboxyl, amide, carbonyl or ester groups). Reso-nances for proteins also appear between 1.5 and2.8 ppm. The signals covering the 3.2–4.5 ppmrange include contributions from H on Ca to anoxygen or nitrogen atom (Chen et al., 2000; Adaniand Ricca, 2004; Kovac et al., 2004). Protonsattached to aminomethine and/or methylene groupsbonded to amide functional groups show resonanceat 3.1–3.35 ppm (Montoneri et al., 2003), althoughmore intense signals appear at 3.7–3.9 ppm, attrib-uted to CHOH and CH2OH groups. In the case ofcomposts, these functional groups may indicatethe presence of methoxyphenylpropyl repeatingunits which typically occur in lignin, and/or the

4000 3500 3000 2500 2000 1500 1000 500

STW

OVC

FM

FMC

OLV

OLVC

GW

GWC

DW

DWC

ν (cm-1)

2850

2925

1715

16301120

1045

3430 1570

1400

Fig. 3. FTIR spectra of humic extracts from non-composted andcomposted organic materials.

M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023 2019

presence of polysaccharide moieties (Adani et al.,2006).

In the case of GWC, OLVC and OVC, an increasein Hal and Har and a decrease in Ha content isobserved when compared with GW, OLV andSTW, respectively. The loss of Ha percentage andthe increase in aromatic protons may be related tothe corresponding decrease in O-alkyl C and increasein aromatic C observed by 13C NMR. On the otherhand, FMC and DWC showed a decrease in aliphaticprotons and an increase in Ha and Har, supported bythe changes observed by 13C NMR (Table 3).

Comparison of proton and carbon aromaticitieshas been reported to be useful (Lee et al., 1998; Wil-son et al., 1999; Peuravuori, 2005; Peuravuori et al.,2006). Thus, high carbon aromaticity and low pro-ton aromaticity would reflect a high degree of aro-matic ring substitution or condensation. With thisin mind, HarH/CarC ratios were calculated (Table3, H/C is the hydrogen/carbon ratio). Fulvic acidsshowed greater values than humic acids, indicatingthat to some extent humic acids possess more sub-stituents on the aromatic rings and/or more con-densed aromatic structures than fulvic acids. Theresults also showed that humic and fulvic acids pre-sented a higher degree of aromatic carbon substitu-tion and/or condensed aromatic rings whencompared with the organic substances extractedfrom composted and non-composted materials.

This ratio did not have a clear trend in com-posted materials, experiencing a decrease in GWCand OVC but an increase in DWC, FMC andOLVC. In some cases the value was higher than 1.The reason for this is that the calculation of Car

and Har necessarily includes non-aromatic C@CAHresonances, and this is specially evident in the caseof AHA, STW and GW, that yielded HarH/CarCratios higher than 1.

3.4. FTIR spectroscopy

The FTIR spectra of the THE from compostedmaterials exhibited the same peaks as those extractedfrom fresh organic materials, but these spectradiffered in the relative intensity of some bands(Fig. 3). The broad band centered at around 3400cm�1 corresponds to O–H stretching of hydroxylgroups of alcohols, phenols and organic acids, aswell as N–H groups; peaks at 2925 and 2850 cm�1

are caused by symmetric and asymmetric stretchingvibrations of C–H in CH2 and CH3 groups. Theband (a shoulder in some cases) at 1715 cm�1 is

attributed to the C@O stretching vibration ofCOOH, ketones, aldehydes and esters. The bandcentered at around 1640–1620 cm�1 may be relatedto aromatic C@C stretching and C@O stretching ofquinone and/or conjugated ketone and amidegroups (amide I). The shoulder appearing at 1460–1450 cm�1 is generated by aliphatic C–H deforma-tions and aromatic ring vibrations. The peak at1400–1390 cm�1 is attributed to O–H deformation,C–O stretching of phenolic OH and C–H deforma-tion of CH2 and CH3 groups. A weak band between1260 and 1220 cm�1 is produced by amides orethers, and a broad band between 1120 and980 cm�1 with a sharp peak centered near1045 cm�1 is related to C–O stretching of polysac-charide or polysaccharide-like substances, as well

CHHA

CZHA

AHA

LSHA

PRHA

SRFA

WRFA

2850

2925

1715

1620 1240

1040

3430

1400

4000 3500 3000 2500 2000 1500 1000 500

ν (cm-1)

Fig. 4. FTIR spectra of HAs and FAs from soils and IHSS.

2020 M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023

as silicate impurities. Bands at 1126 and 1045 cm�1

are also characteristic of aromatic C–H in-plaindeformation for syringyl and guaiacyl alcohols,two structural components of lignin (Stevenson,1994; Pretsch et al., 1998; Sun and Tomkinson,2002; Amir et al., 2004; Ait Baddi et al., 2004a,b).

In general, transformations that occurred duringthe different composting processes were reflected bya decrease in the bands at 2925–2850 cm�1 (exceptfor FMC) and at 1040 cm�1, and an increase inthose at 1715, 1640, 1460 and 1400 cm�1. The risein these bands suggests an increase in carbonylgroups (COOH, ketones, aldehydes, esters) as wellas aromatic, phenolic and quinone structures. Thesechanges observed by FTIR indicate the decrease,during composting, in aliphatic and polysaccharidestructures and the increase in more oxidized and,probably, polycondensed aromatic components. Ingeneral, these results are in line with those obtainedin the 13C NMR study.

Humic and fulvic acids have similar spectra(Fig. 4), the main difference being that the intensityof the 1715 cm�1 band is considerably stronger infulvic acids because of the occurrence of moreCOOH groups. As shown by 13C NMR spectra, ful-vic acids have greater content in carbonyl C than inaromatic C.

3.5. HPSEC

In this study, no attempt was made to determinethe absolute molecular weight of humic substances.Chromatograms were only used for relative com-parison of molecular size distributions between thedifferent systems.

As can be seen in Fig. 5, the chromatograms pre-sented different profiles between humic or fulvicacids extracted from soils, THE from compostedmaterials and those from fresh organic raw mixture.However, samples belonging to the same groupshowed a similar pattern.

THE from composted materials showed an elu-tion profile corresponding to higher molecular sizesthan those corresponding to THE from non-com-posted materials. Several authors (Sanchez-Mone-dero et al., 2002) have also observed an increase inthe average molecular weight of humic substancesextracted from different composts, suggesting thatthis could be the result of condensation reactionsof different fractions to the humic ‘‘core’’ producingmacromolecules, or to the degradation of the small-est fractions and the consequent enrichment of the

largest ones. Other authors attribute this to biodeg-radation followed by the formation of more poly-condensed humic structures (Jouraiphy et al., 2005).

3.6. Relation between UV–Vis and fluorescence

parameters and 13C NMR, 1H NMR, elemental

analysis and FTIR

As for the relationships between the parametersderived from the UV–Visible and fluorescence stud-ies (Fuentes et al., 2006) (Table 3) and the structuralfeatures derived from this study (Table 3), differentgeneral patterns were observed depending on theorganic systems included in the comparison. Thus,when all organic systems (soil humic acids, IHSSstandards, THE from composted and from freshmaterials) are considered, a significant positive cor-relation (P < 0.05, data not shown) was observedbetween UV–Visible and fluorescence indexes (e280,e600, EET/EBz, A440 and A4/A1, defined in Table 3

0 2 6 10 11 12 13 14 15

DWC

DW

LSHA

WRFA

OLV

OLVC

Ve (mL)

543 9871

Fig. 5. HPSEC chromatograms (recorded with the refractiveindex detector) of a humic acid (LSHA), a fulvic acid (WRFA),and the humic extracts from two raw initial mixtures (DW andOLV) and their corresponding composted material (DWC andOLVC).

M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023 2021

footnote) and % total C, C/H ratio, and the percent-ages of aromatic C and phenolic C. These resultswere also associated with significant negative corre-lations between the UV–Visible and fluorescenceindexes and % H, % O, O/C ratio and, in somecases, with carboxylic C. These results seem to indi-cate that humification is associated with both morearomatic rings in the structure with the presence ofphenol groups, and less oxygen containing func-tional groups, principally carboxylic groups.Regarding the possibility that humification is alsorelated with more condensation in the aromaticmoieties, our results do not permit us to assess thisquestion. In fact no significant correlations betweenthe HarH/CarC ratio and the different UV–Visibleand fluorescence parameters were observed.

However, when only THE from composted andfresh materials are considered, in general the samepatterns (the main difference consisted of anincrease in the carboxylic C with composting) areobserved, except in the case of FM–FMC andDW–DWC systems (Table 3). In these systems,

the general increase in the values of UV–Visibleand fluorescence indices with composting was notcorrelated with increases in the aromatic characteror the presence of phenol and carboxylic groups.This result indicates that other structural featuresin addition to those related to the total aromaticcharacter must be related to the increase in UV–Vis-ible and fluorescence indices. This structural prop-erty could be related to the condensation degree inaromatic rings and/or the conjugation degree inaromatic and aliphatic moieties.

In summary, these results seem to indicate twodifferent phases associated with humification. A firstphase consisting of the first transformation of freshplant and microbial material into more humifiedmaterials, that could be represented in this studyby THE from fresh and composted materials, inwhich the humification associated with compostingis related to increases in the aromatic characterand the presence of more acidic functional groupsin the structure. This process seems to be also asso-ciated with the development of molecular fractionswith larger sizes. THE from composted materialspresented some structural features and values ofthe UV–Visible and fluorescence indexes similar tothose of IHSS reference fulvic acids. However, theresults obtained also indicate that more advancedstages of humification involve increases in boththe aromatic character that could be linked to ahigher degree of polycondensation, and the presenceof phenol groups; and a decrease in carboxylicgroups that might be related to aromatic condensa-tion processes producing aromatic quinone-typestructures.

4. Conclusions

Humic extracts from composted materialsyielded higher C/H and lower C/N ratios thanhumic extracts from their initial fresh organic mix-tures. These changes could be the result of the deg-radation of carbohydrates, polysaccharides or fattyacids, and the incorporation into the humic ‘‘core’’of N containing groups through condensation ofproteins and modified lignin, or sugar-amine con-densation. FTIR and 13C NMR spectroscopiesshowed a decrease in aliphatic content and an incre-ment in polar functional groups (O- or N-contain-ing functional groups) as a result of composting.When compared with FA and HA, THE from com-posted materials showed more structural similaritieswith FA than with HA.

2022 M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023

These results support the usefulness of e280,EET/EBz and A440 parameters as indicators of thedegree of evolution (humification) of the compostedmaterials. e280 reflects the aromatic content, whileEET/EBz ratio seems to be an indicator of the degreeof substitution in the aromatic ring with polarfunctional groups, as the results from FTIR and13C NMR suggest. Likewise, A440 parameter maybe related to the degree of complexity in the aromaticmoiety.

Finally, these results also indicate that humifica-tion seems to be associated with both an increase inthe aromatic character of the system with the pres-ence of phenol groups as principal substituents,and a reduction in oxygen containing functionalgroups principally carboxylic groups. Likewisesome of these results indicate that increases in aro-matic condensation and the degree of conjugationcould be also involved during humification. Morestudies, therefore, are needed in order to elucidatethese questions. In particular, specific 13C NMRand pyrolysis-MS studies could prove very useful.

Acknowledgements

This research was founded by the Roullier Groupand the Government of Navarra. Special thanks toDavid Rhymes for kindly improving the English ofthe manuscript.

Associate Editor—Ian D. Bull

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