- Email: [email protected]
The complementary use of 1H NMR, 13C NMR, FTIR and size exclusion chromatography to investigate the principal structural changes associated with composting of organic materials with diverse origin
Available online at www.sciencedirect.com
Organic Geochemistry Organic Geochemistry 38 (2007) 2012–2023 www.elsevier.com/locate/orggeochem
The complementary use of 1H NMR, 13C NMR, FTIR and size exclusion chromatography to investigate the principal structural changes associated with composting of organic materials with diverse origin Marta Fuentes a, Roberto Baigorri b, Gustavo Gonza´lez-Gaitano a, Jose´ Ma Garcı´a-Mina a,b,* a
Department of Chemistry and Soil Chemistry, University of Navarra, 31080 Pamplona, Spain b CIPAV-Roullier Group, Inabonos, Polı´gono Arazuri-Orcoyen, 31160 Orcoyen, Spain
Received 5 December 2006; received in revised form 27 July 2007; accepted 23 August 2007 Available 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 mixtures were 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 acids and two reference fulvic acids (1S104H, 1R103H, 1R101F and 1R107F) obtained from the International Humic Substances Society were also characterized using these techniques, in order to compare the features of reference humic and fulvic acids with those of composted materials. Likewise, the results were compared with those obtained in previous studies, in which UV–Visible and fluorescence spectroscopies were employed to characterize the humification degree of the molecular systems. The results obtained by 13C CPMAS NMR, 1H NMR and FTIR indicate that, in general, humification seems to be associated with an increase in the aromatic character of the systems, with the presence of phenol groups as principal substituents and a reduction in oxygen containing functional groups, principally carboxylic or carbonylic groups, as well as the development of molecular fractions with larger size. These results also support the suitability of UV–Visible and fluorescence spectroscopies in the assessment of the humification course of humic extracts in composting processes. Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction *
Corresponding author. Address: Department of Chemistry and Soil Chemistry, University of Navarra, 31080 Pamplona, Spain. Tel.: +34 948324550; fax: +34 948324032. E-mail address: [email protected] (J.M. Garcı´a-Mina).
Recycling organic wastes from industrial and domestic sources by means of composting has arisen in recent years as an alternative to classical soil
0146-6380/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2007.08.007
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
amendment (Gonza´lez-Vila et al., 1999; Ait Baddi et al., 2004a,b; Amir et al., 2004). These organic residues have to be pretreated in order to stabilize them and to reduce their initial toxicity. Non-composted residues or immature composts applied to agricultural soils degrade rapidly, and may cause phytotoxicity due to the presence of simple organic acids, and decreases in oxygen and nitrogen concentrations, among other harmful effects on soil properties (Mustin, 1987; Jime´nez and Garcı´a, 1992). The content in potential organic (i.e., polycyclic aromatic hydrocarbons) and inorganic (heavy metals) pollutants of composts must also be controlled, as these contaminants may migrate to groundwater or accumulate in plants (Oleszczuk and Baran, 2005; Alvarenga et al., 2007; Oleszczuk, in press). The quality and stability of composts depend largely on the properties of the initial materials, the time of composting and the oxidative conditions of treatment that influence microbial activities, resulting in different degrees of degradation and transformation of the initial organic mixtures. Composting has been described as an accelerated version of the decomposition processes naturally occurring in the soil (Ait Baddi et al., 2004a). Therefore, the characterization of the changes in the composition of the compost and its comparison with reference humic substances generated in soils are two important points to consider when assessing the quality and properties of the final products, and studying the singular structural changes and features associated with humification. These changes have classically been attributed to increases in aromatic content in humic substances, although other studies have shown that accumulation of recalcitrant aliphatic structures also takes place during composting (Almendros et al., 2000). Organic matter transformation during composting can be estimated by C/N ratios, soluble organic carbon concentration in water extracts or humic acid to fulvic acid ratio (Hsu and Lo, 1999; Jouraiphy et al., 2005). Indices derived from fluorescence and UV–Visible spectroscopies have also proven to be useful in evaluating the humification degree of HS extracted from organic materials of diverse origin (Korshin et al., 1997; Peuravuori and Pihlaja, 2002; Milori et al., 2002; Fuentes et al., 2006), as well as Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopies (NMR) (Gonza´lez-Vila et al., 1999; Sa´nchez-Monedero et al., 2002; Ait Baddi et al., 2004a; Amir et al., 2004; Jouraiphy et al., 2005).
2013
In a previous study (Fuentes et al., 2006), extracts obtained with alkaline solutions from several composted materials and from their initial raw organic mixtures, in addition to various humic and fulvic acids from soils, including International Humic Substances Society (IHSS) reference materials, were studied using UV–Visible and fluorescence spectroscopies. Several indices indicated reasonably well the evolution (humification) of the different systems, indicating that humification might be related to: (i) significant changes in the aromatic character of the samples, which in turn could be associated with more functional ring substitution and polycondensation; and (ii) significant changes in the molecular size distribution throughout humification. In order to study more in depth these possible structural changes associated with humification, we have investigated the same organic systems characterized by UV–Visible and fluorescence spectroscopies (Fuentes et al., 2006) through the complementary use of elemental analysis, 1H NMR, 13C NMR, FTIR and high pressure size exclusion chromatography (HPSEC). Likewise, this study has permitted us to compare the properties of composted materials with those of humic and fulvic acids extracted from soils. 2. Materials and methods 2.1. Organic materials The organic systems studied were divided into three groups: (i) humic substances (humic and fulvic acids) with diverse origin, including IHSS standards; (ii) organic substances contained in alkaline extracts obtained from composted materials; and (iii) organic substances contained in alkaline extracts obtained from the initial organic materials used in the different composting procedures. Soil humic substances were isolated from soil samples of different locations: China (CHHA) and Czech Republic (CZHA). Commercial humic acid was 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 composted and non-composted raw materials were defined 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 initial mill olive residues (OLV); compost of domestic wastes (DWC) and initial domestic wastes (DW); compost of ovine manures plus straw (OVC) and straw (STW); compost of a mixture of animal manures (FMC) and the initial mixture (noncomposted) of animal manures (FM). The characterization of these materials is summarized in Table 1. The starting materials were piled in rows, and these piles were periodically turned over in order to aerate and homogenize the mixtures. The time of composting was four months, and during this period piles were turned six times.
by a LECO CHN 900 analyser. The oxygen content was determined by difference (ash-free basis). 2.4.
13
C NMR spectroscopy
13
C NMR spectra were obtained on a Varian Unity 300 spectrometer at 75.429 MHz using the cross-polarization magic angle spinning technique (CPMAS), with a spinning speed of 5 kHz, 90° pulse width, 69 ms acquisition time and 1.0 s delay. 2.5. 1H NMR spectroscopy Solution 1H NMR spectra were recorded on a Varian Unity-300 spectrometer at 300 MHz, using a 5 mm multinuclear probe, with 90° pulse angle, a sweep width of 4000 Hz and a line broadening of 0.5 Hz. Gated irradiation was applied between acquisitions 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 shift standard.
2.2. Extraction procedure The different organic systems were extracted from solid samples of the organic materials with 0.1 M NaOH (24 h of mechanical shaking in darkness) at 22 °C. The air was displaced by N2 in the extracts in order to avoid possible oxidation during the extraction process. The sample:extractant ratio used was 1:6 for all samples but 1:10 for straw (this needed a larger volume of NaOH solution because of its great liquid absorption capacity). The suspension was then centrifuged at 11,100 g for 15 min and the alkaline supernatants were treated with an acidic-cation exchange resin (Amberlite IR-120H, Aldrich) in order to lower the pH to 3.5 before freeze-drying.
2.6. FTIR spectroscopy Pellets were prepared by mixing 1 mg of each freeze-dried sample with 100 mg of KBr so that the mixture became homogeneous. Infrared spectra were recorded on these pellets with a Nicolet Magna-IR 550 spectrometer over the 4000–400 cm 1 range.
2.3. Elemental analysis
2.7. HPSEC study
The carbon, hydrogen and nitrogen contents of the lyophilized samples were analysed in duplicate
Molecular size distribution for humic materials was evaluated by high performance size exclusion
Table 1 Moisture, ash content and elemental composition (w/w %) of the initial organic mixtures and the corresponding composted materials Organic substances
Moisturea
Ashb
Cc
Hc
Nc
Oc
C/N
C/H
Ovine manure
STW OVC
8.9 8.0
0.6 7.0
48.2 51.3
5.8 5.3
0.3 4.3
45.6 39.1
177.9 14.0
0.69 0.81
Animal manure
FM FMC
8.0 4.6
11.1 10.7
57.4 53.3
6.2 6.2
8.0 7.2
28.4 33.4
8.4 8.7
0.77 0.72
Olive wastes
OLV OLVC
9.1 8.4
2.5 4.0
54.0 56.2
6.5 5.2
1.5 2.6
38.0 36.0
41.3 25.5
0.69 0.90
Grape wastes
GW GWC
9.5 12.0
3.1 5.5
52.4 58.0
5.7 5.1
1.9 3.7
40.0 33.2
32.7 18.3
0.77 0.94
Domestic wastes
DW DWC
7.2 8.4
2.7 7.4
43.2 54.5
4.6 5.6
1.3 4.8
50.9 35.1
38.7 13.3
0.78 0.82
a b c
Wet matter. Dry matter. Dry and ash-free matter.
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
2015
3.1. Elemental analysis
chromatography. The chromatographic system consisted of a Waters 600 Controller pump followed by two detectors in series: a Waters 996 Photodiode Array Detector set at 400 nm, and a Waters 2424 refractive index detector (RI). Size exclusion separation occurred through a PL Aquagel-OH 30 column (Polymer Laboratories), preceded by a guard column with the same stationary phase. The overall molecular weight range of separation for this column is 100–300,000 Da. For each sample, solutions of 800 mg of organic carbon l 1 were prepared in 0.05 M NaNO3. The injection volume of all samples was 100 ll, the eluent used was 0.05 M NaNO3 (pH 7), and the flow rate was 1 ml/min. Void volume (V0 = 6.65 ml) and permeation volume (Vp = 11.82 ml) were determined with polyethylene oxide of MW of 43,250 Da and methanol, respectively.
The HA studied (CHHA, CZHA, AHA, LSHA, PRHA) presented the highest content in carbon, around 55–60% (Table 2). Reference fulvic acids showed slightly lower values, but they had higher oxygen content, mainly due to the larger content in COOH groups in those compounds as shown by 13C NMR (Table 3). Values for O/C atomic ratios fell within the range described in the literature for HA and FA. Thus, HAs presented O/C values around 0.5, whereas those of FAs were close to 0.7 (Stevenson, 1994). Differences in the nitrogen content were not significant, although C/N ratio varied, being lower in HAs and indicating larger nitrogen fixation in these substances. As for the organic substances extracted from composted and non-composted materials, the carbon content decreased in OVC, OLVC and FMC, but increased in GWC and DWC. The percentage of H and C/N ratios decreased in all cases. The C/H ratios increased in all cases to varying degrees, while the N content increased in all cases except FMC. The increase in the C/H ratio suggests that changes in chemical composition during the composting
3. Results and discussion The results obtained using the different techniques will be presented and commented upon separately, and afterwards these results will be related to those previously obtained using UV–Vis and fluorescence spectroscopies as described in Fuentes et al. (2006).
Table 2 Elemental analysis for HAs and FAs from soils and IHSS and for humic-like substances extracted from raw initial organic mixtures and composts (see acronyms in the text, Section 2.1) Families
Organic substances
Elemental analysis %C
%H
%N
%O
O/C
HS
CHHA CZHA AHA
59.6 59.4 53.4
1.6 2.4 3.7
1.4 1.3 0.7
37.4 36.8 42.2
0.47 0.46 0.59
49.7 53.3 89.0
3.10 2.06 1.20
HS-IHSS
LSHA PRHA SRFA WRFA
62.2 55.7 52.5 53.5
3.6 3.8 4.3 4.2
1.2 3.6 0.7 1.1
30.5 36.9 43.5 41.7
0.37 0.50 0.62 0.58
60.5 18.0 87.5 56.7
1.44 1.22 1.02 1.06
Ovine manure
STW OVC FM FMC
40.1 33.7 43.2 34.2
3.7 3.0 4.4 3.7
0.6 3.7 10.0 7.7
55.5 59.5 42.4 54.5
1.04 1.32 0.74 1.19
78.0 10.6 5.0 5.2
0.90 0.94 0.82 0.78
Olive wastes
OLV OLVC
56.0 48.7
5.0 4.1
0.6 2.5
38.4 44.7
0.51 0.69
108.9 22.7
0.93 0.99
Grape wastes
GW GWC
38.9 48.1
3.9 3.2
1.0 4.6
56.3 44.1
1.08 0.68
45.4 12.2
0.83 1.25
Domestic wastes
DW DWC
39.1 41.4
5.1 3.3
1.4 7.1
54.4 48.2
1.04 0.78
32.6 6.8
0.63 1.04
Animal manure
Atomic ratios C/N
C/H
2016 Table 3 Relative abundances of different carbon and hydrogen types (in %) measured by 13C CPMAS NMR and 1H NMR spectroscopy, and parameters obtained by UV–Vis and fluorescence spectroscopies Families
Organic substances
13
1 H NMR (Chemical shift range in ppm)
C NMR (Chemical shift range in ppm) 45–110
110–160
140–160
160–215
0.5–3
3–4.5
6–8.5
HarH/CarC
EET =EBz b
e280 c
e600 d
A440e
Alkyl C
O-alkyl C
Aromatic C
Phenolic C
Carbonylic C
Hal
Ha
Har
HS
CHHA CZHA AHA
13.8 27.4 62.5
3.2 12.6 18.8
79.5 46.5 12.1
5.4 12.8 5.3
3.5 13.5 6.6
24 70 42
53 17 16
23 13 42
0.09 0.13 3.03
0.90 0.86 0.84
980 1064 777
92.5 51.9 50.6
10230 16160 8110
HS-IHSS
LSHA PRHA SRFA WRFA
26.6 20.1 32.5 23.2
14.9 10.9 14.2 12.2
48.7 37.3 15.6 29.0
12.8 12.4 5.0 9.8
9.8 31.7 37.7 35.6
31 52 71 37
68 40 22 36
1 8 7 27
0.01 0.18 0.47 0.88
0.82 0.77 0.62 0.67
1402 834 449 500
80.1 58.3 46.5 16.3
8540 5970 4490 4750
Ovine manure
STW OVC
18.6 17.7
49.7 25.0
8.2 22.3
3.3 10.0
23.5 35
26 48
65 46
9 6
1.21 0.26
0.45 0.50
116 373
2.0 14.7
1010 4150
Animal manure
FM FMC
33.7 30.4
13.8 24.0
11.8 9.6
4.0 3.0
40.7 36
72 27
24 68
4 5
0.45 0.68
0.41 0.56
108 286
3.0 9.8
2040 4110
Olive wastes
OLV OLVC
23.2 22.2
56.4 29.3
11.2 19.7
5.4 7.1
9.2 28.8
40 45
58 47
2 8
0.23 0.42
0.47 0.65
106 256
13.2 16.9
1840 4550
Grape wastes
GW GWC
3.5 21.6
46.9 13.1
1.5 26.8
1.0 10.0
48.1 38.5
28 56
70 43
2 1
1.33 0.15
0.33 0.76
55 415
3.1 19.5
780 4480
Domestic wastes
DW DWC
23.7 25.4
28.2 34.9
20.3 16.4
9.5 5.0
27.8 23.3
61 56
36 39
3 5
0.26 0.32
0.51 0.62
70 354
2.8 26.4
680 3830
a b c d e
Values presented for EET/EBz, e280, e600 and A440 are taken from a previous study (Fuentes et al., 2006). Ratio of absorbances at 253 and 220 nm in the UV spectrum. Molar absorptivity at 280 nm (L cm 1 mol 1 of organic carbon). Molar absorptivity at 600 nm (L cm 1 mol 1 of organic carbon). Area under fluorescence emission spectra (460–650 nm) with kexc = 440 nm.
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
0–45
UV–Vis and fluorescence parametersa
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
process could be due to the degradation of substances with low C/H, such as carbohydrates, polysaccharides or fatty acids, along with an increase in unsaturated structures relative to saturated ones (Miiki et al., 1997; Sa´nchez-Monedero et al., 2002). Nitrogen compounds are thought to be incorporated to the humic ‘‘core’’ through condensation of proteins and modified lignin, reactions between N-containing compounds and quinones derived from lignin, or sugar-amine condensation (Stevenson, 1994). The absence of significant differences in the chemical composition corresponding to the organic substances extracted from fresh and composted materials has also been found by other authors (Riffaldi et al., 1983; Inbar et al., 1990; Ait Baddi et al., 2004a), who reported that the chemical composition of humic-like substances extracted from various organic sources at different stages of maturity remained almost unchanged during composting. On the other hand, the organic substances extracted from composted materials presented C/H ratio values similar to those of fulvic acids and lower than those of humic acids. These results could indicate the presence of less aromatic structures in the organic substances extracted from composted materials compared to the soil humic acids. Finally, the high nitrogen values found in the organic substances extracted from composted materials could be indicative of high contents of non-humified biomolecules (such as polysaccharides and polypeptides) or to the incomplete hydrolysis of proteinaceous constituents in these systems (Sa´nchez-Monedero et al., 2002; Ait Baddi et al., 2004a). 3.2.
13
C NMR study
In general the main trends inferred from the elemental analysis study were confirmed by the results obtained in the 13C NMR study. The 13C NMR spectra are shown in Fig. 1. For data analysis, spectra were divided into chemical shift regions assigned to the following classes of chemical 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 carbonyl C (160–220 ppm). The relative intensity of these regions was determined by the integration of the corresponding peak areas (Malcolm, 1989; Stevenson, 1994; Gonda et al., 2005).
2017
Significant changes were observed between GWC, OLVC, OVC and their respective initial fresh organic materials GW, OLV, STW. In the three cases the aliphatic content decreased due to a loss of alkyl carbon bonded to oxygen, whereas percentages of aromatic, phenolic, and carbonylic C increased (Table 3). These changes may reflect that during composting, the unstable organic compounds such as aliphatic materials are transformed through intense microbial activities into more stable humic compounds with more oxidized, olefinic or aromatic structures that could include more polycondensed rings (Amir et al., 2004). In the case of DW–DWC and FM–FMC systems, however, the distribution of functional groups did not undergo important variations, which mainly consisted of an increase in aliphatic C (mainly O-alkyl C) and a slight decrease in aromatic and carbonylic C (Table 3). Gonza´lez-Vila et al. (1999) studied composts from urban wastes and did not distinguish a clear progressive trend of the organic components in the course of the composting process. Composting led to simultaneous degradation of all types of C, with no selective accumulation of any preferentially stable forms. Almendros et al. (2000) found that in most cases for composted forest and shrub biomass, the recalcitrant material accumulated during composting was not exclusively aromatic in nature and that the presence of tannins may contribute, through their selective preservation and condensation reactions, to limiting the decomposition of aliphatic biomacromolecules. Likewise, these authors indicated that the decrease in the carboxylic content might be due to the degradation of lipids. These authors also emphasize the possible interfering signals of protein moieties in the humic substances extracted from composts, as amino acids contribute to resonance in the alkyl C, Oalkyl C and carbonyl C regions. On the other hand, soil humic acids showed lower aliphatic content (mainly methyl and methylene groups) and higher aromatic content when compared with the organic substances extracted from composted materials, except in the case of the Aldrich humic acid (AHA) that was mostly aliphatic, in agreement with what had been previously reported (Malcolm and MacCarthy, 1986; Shin et al., 1999). It was noteworthy the similarities in functional groups between the organic substances extracted from composted materials and the IHSS fulvic acids.
2018
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
a
b STW
CHHA OVC FM
CZHA
FMC AHA OLV LSHA
OLVC
PRHA
GW GWC
SRFA DW WRFA
DWC 225 200 175 150 125 100 75
50
25
0
225 200 175 150 125 100 75
50
25
0
δ (ppm)
δ (ppm)
Fig. 1. 13C CPMAS NMR spectra of: (a) HAs and FAs from soils and IHSS; and (b) humic extracts from non-composted and composted organic materials.
3.3. 1H NMR study Several representative 1H NMR spectra recorded for the different systems studied are shown in Fig. 2. Proton spectra are typically divided into three main regions: (i) 0.5–3 ppm, resonance of alkyl protons and protons attached to carbon in a to aromatic
PRHA D2O peak
WRFA
OLV
OLVC 8
6
4
2
0
δ (ppm) 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 oxygen or nitrogen (Ha); and (iii) 6–8.5 ppm, protons attached to unsaturated carbons and aromatic protons (Har) (Lambert and Lankes, 2002). The integration of these areas and the percentage of each type of proton are shown in Table 3. In the region of resonance of aliphatic protons, peaks around 0.9 ppm are assigned to protons of terminal methyl groups, whereas signals between 1.2 and 1.8 ppm are attributed to methylene and methine protons. The peaks appearing in the range of 2.0–2.8 ppm can be assigned to protons adjacent to functional groups with electronegative atoms (carboxyl, amide, carbonyl or ester groups). Resonances for proteins also appear between 1.5 and 2.8 ppm. The signals covering the 3.2–4.5 ppm range include contributions from H on Ca to an oxygen or nitrogen atom (Chen et al., 2000; Adani and Ricca, 2004; Kovac et al., 2004). Protons attached to aminomethine and/or methylene groups bonded to amide functional groups show resonance at 3.1–3.35 ppm (Montoneri et al., 2003), although more intense signals appear at 3.7–3.9 ppm, attributed to CHOH and CH2OH groups. In the case of composts, these functional groups may indicate the presence of methoxyphenylpropyl repeating units which typically occur in lignin, and/or the
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
presence of polysaccharide moieties (Adani et al., 2006). In the case of GWC, OLVC and OVC, an increase in Hal and Har and a decrease in Ha content is observed when compared with GW, OLV and STW, respectively. The loss of Ha percentage and the increase in aromatic protons may be related to the corresponding decrease in O-alkyl C and increase in aromatic C observed by 13C NMR. On the other hand, FMC and DWC showed a decrease in aliphatic protons and an increase in Ha and Har, supported by the changes observed by 13C NMR (Table 3). Comparison of proton and carbon aromaticities has been reported to be useful (Lee et al., 1998; Wilson et al., 1999; Peuravuori, 2005; Peuravuori et al., 2006). Thus, high carbon aromaticity and low proton aromaticity would reflect a high degree of aromatic ring substitution or condensation. With this in mind, HarH/CarC ratios were calculated (Table 3, H/C is the hydrogen/carbon ratio). Fulvic acids showed greater values than humic acids, indicating that to some extent humic acids possess more substituents on the aromatic rings and/or more condensed aromatic structures than fulvic acids. The results also showed that humic and fulvic acids presented a higher degree of aromatic carbon substitution and/or condensed aromatic rings when compared with the organic substances extracted from composted and non-composted materials. This ratio did not have a clear trend in composted materials, experiencing a decrease in GWC and OVC but an increase in DWC, FMC and OLVC. 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@CAH resonances, and this is specially evident in the case of AHA, STW and GW, that yielded HarH/CarC ratios higher than 1. 3.4. FTIR spectroscopy The FTIR spectra of the THE from composted materials exhibited the same peaks as those extracted from fresh organic materials, but these spectra differed in the relative intensity of some bands (Fig. 3). The broad band centered at around 3400 cm 1 corresponds to O–H stretching of hydroxyl groups of alcohols, phenols and organic acids, as well as N–H groups; peaks at 2925 and 2850 cm 1 are caused by symmetric and asymmetric stretching vibrations of C–H in CH2 and CH3 groups. The band (a shoulder in some cases) at 1715 cm 1 is
2019
3430
1570 2925
1630 1400
2850
1120 1045
1715
STW
OVC
FM
FMC
OLV
OLVC
GW
GWC
DW
DWC
4000
3500
3000
2500
2000
1500
1000
500
ν (cm-1)
Fig. 3. FTIR spectra of humic extracts from non-composted and composted organic materials.
attributed to the C@O stretching vibration of COOH, ketones, aldehydes and esters. The band centered at around 1640–1620 cm 1 may be related to aromatic C@C stretching and C@O stretching of quinone and/or conjugated ketone and amide groups (amide I). The shoulder appearing at 1460– 1450 cm 1 is generated by aliphatic C–H deformations and aromatic ring vibrations. The peak at 1400–1390 cm 1 is attributed to O–H deformation, C–O stretching of phenolic OH and C–H deformation of CH2 and CH3 groups. A weak band between 1260 and 1220 cm 1 is produced by amides or ethers, and a broad band between 1120 and 980 cm 1 with a sharp peak centered near 1045 cm 1 is related to C–O stretching of polysaccharide or polysaccharide-like substances, as well
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-plain deformation 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 during the different composting processes were reflected by a decrease in the bands at 2925–2850 cm 1 (except for FMC) and at 1040 cm 1, and an increase in those at 1715, 1640, 1460 and 1400 cm 1. The rise in these bands suggests an increase in carbonyl groups (COOH, ketones, aldehydes, esters) as well as aromatic, phenolic and quinone structures. These changes observed by FTIR indicate the decrease, during composting, in aliphatic and polysaccharide structures and the increase in more oxidized and, probably, polycondensed aromatic components. In general, these results are in line with those obtained in the 13C NMR study. Humic and fulvic acids have similar spectra (Fig. 4), the main difference being that the intensity of the 1715 cm 1 band is considerably stronger in fulvic acids because of the occurrence of more COOH groups. As shown by 13C NMR spectra, fulvic acids have greater content in carbonyl C than in aromatic C.
3430
In this study, no attempt was made to determine the absolute molecular weight of humic substances. Chromatograms were only used for relative comparison of molecular size distributions between the different systems. As can be seen in Fig. 5, the chromatograms presented different profiles between humic or fulvic acids extracted from soils, THE from composted materials and those from fresh organic raw mixture. However, samples belonging to the same group showed a similar pattern. THE from composted materials showed an elution profile corresponding to higher molecular sizes than those corresponding to THE from non-composted materials. Several authors (Sa´nchez-Monedero et al., 2002) have also observed an increase in the average molecular weight of humic substances extracted from different composts, suggesting that this could be the result of condensation reactions of different fractions to the humic ‘‘core’’ producing macromolecules, or to the degradation of the smallest fractions and the consequent enrichment of the
1620 2850
1240
1715 1400
1040
CHHA
CZHA
AHA
LSHA
PRHA
SRFA
WRFA
4000
3.5. HPSEC
2925
3500
3000
2500
2000 ν (cm-1)
1500
1000
500
Fig. 4. FTIR spectra of HAs and FAs from soils and IHSS.
largest ones. Other authors attribute this to biodegradation followed by the formation of more polycondensed 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 parameters derived from the UV–Visible and fluorescence studies (Fuentes et al., 2006) (Table 3) and the structural features derived from this study (Table 3), different general patterns were observed depending on the organic systems included in the comparison. Thus, when all organic systems (soil humic acids, IHSS standards, THE from composted and from fresh materials) are considered, a significant positive correlation (P < 0.05, data not shown) was observed between UV–Visible and fluorescence indexes (e280, e600, EET/EBz, A440 and A4/A1, defined in Table 3
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
LSHA
WRFA
DW
DWC
OLV
OLVC 0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
Ve (mL) Fig. 5. HPSEC chromatograms (recorded with the refractive index detector) of a humic acid (LSHA), a fulvic acid (WRFA), and the humic extracts from two raw initial mixtures (DW and OLV) and their corresponding composted material (DWC and OLVC).
footnote) and % total C, C/H ratio, and the percentages of aromatic C and phenolic C. These results were also associated with significant negative correlations between the UV–Visible and fluorescence indexes and % H, % O, O/C ratio and, in some cases, with carboxylic C. These results seem to indicate that humification is associated with both more aromatic rings in the structure with the presence of phenol groups, and less oxygen containing functional groups, principally carboxylic groups. Regarding the possibility that humification is also related with more condensation in the aromatic moieties, our results do not permit us to assess this question. In fact no significant correlations between the HarH/CarC ratio and the different UV–Visible and fluorescence parameters were observed. However, when only THE from composted and fresh materials are considered, in general the same patterns (the main difference consisted of an increase in the carboxylic C with composting) are observed, except in the case of FM–FMC and DW–DWC systems (Table 3). In these systems,
2021
the general increase in the values of UV–Visible and fluorescence indices with composting was not correlated with increases in the aromatic character or the presence of phenol and carboxylic groups. This result indicates that other structural features in addition to those related to the total aromatic character must be related to the increase in UV–Visible and fluorescence indices. This structural property could be related to the condensation degree in aromatic rings and/or the conjugation degree in aromatic and aliphatic moieties. In summary, these results seem to indicate two different phases associated with humification. A first phase consisting of the first transformation of fresh plant and microbial material into more humified materials, that could be represented in this study by THE from fresh and composted materials, in which the humification associated with composting is related to increases in the aromatic character and the presence of more acidic functional groups in the structure. This process seems to be also associated with the development of molecular fractions with larger sizes. THE from composted materials presented some structural features and values of the UV–Visible and fluorescence indexes similar to those of IHSS reference fulvic acids. However, the results obtained also indicate that more advanced stages of humification involve increases in both the aromatic character that could be linked to a higher degree of polycondensation, and the presence of phenol groups; and a decrease in carboxylic groups that might be related to aromatic condensation processes producing aromatic quinone-type structures. 4. Conclusions Humic extracts from composted materials yielded higher C/H and lower C/N ratios than humic extracts from their initial fresh organic mixtures. These changes could be the result of the degradation of carbohydrates, polysaccharides or fatty acids, and the incorporation into the humic ‘‘core’’ of N containing groups through condensation of proteins and modified lignin, or sugar-amine condensation. FTIR and 13C NMR spectroscopies showed a decrease in aliphatic content and an increment in polar functional groups (O- or N-containing functional groups) as a result of composting. When compared with FA and HA, THE from composted materials showed more structural similarities with 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 the degree of evolution (humification) of the composted materials. e280 reflects the aromatic content, while EET/EBz ratio seems to be an indicator of the degree of substitution in the aromatic ring with polar functional groups, as the results from FTIR and 13 C NMR suggest. Likewise, A440 parameter may be related to the degree of complexity in the aromatic moiety. Finally, these results also indicate that humification seems to be associated with both an increase in the aromatic character of the system with the presence of phenol groups as principal substituents, and a reduction in oxygen containing functional groups principally carboxylic groups. Likewise some of these results indicate that increases in aromatic condensation and the degree of conjugation could be also involved during humification. More studies, therefore, are needed in order to elucidate these questions. In particular, specific 13C NMR and pyrolysis-MS studies could prove very useful. Acknowledgements This research was founded by the Roullier Group and the Government of Navarra. Special thanks to David Rhymes for kindly improving the English of the manuscript. Associate Editor—Ian D. Bull References Adani, F., Ricca, G., 2004. The contribution of alkali soluble (humic acid-like) and unhydrolyzed-alkali soluble (corehumic acid-like) fractions extracted from maize plant to the formation of soil humic acid. Chemosphere 56, 13–22. Adani, F., Genevini, P., Tambone, F., Montoneri, E., 2006. Compost effect on soil humic acid: a NMR study. Chemosphere 65, 1300–1307. Ait Baddi, G., Hafidi, M., Cegarra, J., Alburquerque, J.A., Gonza´lvez, J., Gilard, V., Revel, J.-C., 2004a. Characterization of fulvic acids by elemental and spectroscopic (FTIR and 13 C NMR) analyses during composting of olive mill wastes plus straw. Bioresource Technology 93, 285–290. Ait Baddi, G., Alburquerque, J.A., Gonza´lvez, J., Cegarra, J., Hafidi, M., 2004b. Chemical and spectroscopic analyses of organic matter transformations during composting of olive mill wastes. International Biodeterioration and Biodegradation 54, 39–44. Almendros, G., Dorado, J., Gonza´lez-Vila, F.J., Blanco, M.J., Lankes, U., 2000. 13C NMR assessment of decomposition patterns during composting of forest and shrub biomass. Soil Biology and Biochemistry 32, 793–804.
Amir, S., Hafidi, M., Merlina, G., Hamdi, H., Revel, J.-C., 2004. Elemental analysis, FTIR and 13C NMR of humic acids from sewage sludge composting. Agronomie 24, 13–18. Alvarenga, P., Palma, P., Gonc¸alves, A.P., Fernandes, R.M., Cunha-Queda, A.C., Duarte, E., Vallini, G., 2007. Evaluation of chemical and ecotoxicological characteristics of biodegradable organic residues for application to agricultural land. Environment International 33, 505–513. Chen, Y., Katan, J., Gamliel, A., Aviad, T., Snitzer, M., 2000. Involvement of soluble organic matter in increased plant growth in solarized soils. Biology and Fertility of Soils 32, 28– 34. Fuentes, M., Gonza´lez-Gaitano, G., Garcı´a-Mina, J.M., 2006. The usefulness of UV–visible and fluorescence spectroscopies to study the chemical nature of humic substances from soils and composts. Organic Geochemistry 37, 1949– 1959. Gonda, D., Lopez, R., Fiol, S., Antelo, J.M., Arce, F., 2005. Characterization and acid–base properties of fulvic and humic acids isolated from two horizons of an ombrotrophic peat bog. Geoderma 126, 367–374. Gonza´lez-Vila, F.J., Almendros, G., Madrid, F., 1999. Molecular alterations of organic fractions from urban waste in the course of composting and their further transformation in amended soil. The Science of the Total Environment 236, 215–229. Hsu, J.-H., Lo, S.-L., 1999. Chemical and spectroscopic analyses of organic matter transformations during composting of pig manure. Environmental Pollution 104, 189–196. Inbar, Y., Chen, Y., Hadar, Y., 1990. Humic substances formed during the composting of organic matter. Soil Science Society of America Journal 54, 1316–1323. Jime´nez, E.I., Garcı´a, V.P., 1992. Determination of maturity indices for city refuse composts. Agriculture Ecosystems and Environment 38, 331–343. Jouraiphy, A., Amir, S., El Gharous, M., Revel, J.C., Hafidi, M., 2005. Chemical and spectroscopic analysis of organic matter transformation during composting of sewage sludge and green plant waste. International Biodeterioration and Biodegradation 56, 101–108. Korshin, G.V., Li, C.W., Benjamin, M.M., 1997. Monitoring the properties of natural organic matter through UV spectroscopy: a consistent theory. Water Research 31, 1787–1795. Kovac, N., Faganeli, J., Bajt, O., Sket, B., Orel, B., Penna, N., 2004. Chemical composition of macroaggregates in the northern Adriatic Sea. Organic Geochemistry 35, 1095– 1104. Lambert, J., Lankes, U., 2002. Application of Nuclear Magnetic Resonance spectroscopy to structural investigations of refractory organic substances – principles and definitions. In: Frimmel, F.H., Abbt-Braun, G., Heumann, K.G., Hock, B., Lu¨demann, H.-D., Spiteller, M. (Eds.), Refractory Organic Substances in the Environment. Wiley-VCH, Weinheim, pp. 89–95. Lee, G.S.H., Wilson, M.A., Young, B.R., 1998. The application of the ‘‘WATERGATE’’ suppression technique for analyzing humic substances by nuclear magnetic resonance. Organic Geochemistry 28, 549–559. Malcolm, R.L., MacCarthy, P., 1986. Limitations in the use of commercial humic acids in water and soil research. Environmental Science and Technology 20, 904–911.
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023 Malcolm, R.L., 1989. Applications of solid-state 13C NMR spectroscopy to geochemical studies of humic substances. In: Hayes, M.H.B., MacCarthy, P., Malcolm, R.L., Swift, R.S. (Eds.), Humic Substances II: In Search of Structure. Wiley, Chichester, pp. 339–372. Miiki, V., Senesi, N., Ha¨nninen, K., 1997. Characterization of humic material formed by composting of domestic and industrial biowastes. Chemosphere 34, 1639–1651. Milori, D.M.B.P., Martin-Neto, L., Bayer, C., Mielniczuk, J., Bagnato, V.S., 2002. Humification degree of soil humic acids determined by fluorescence spectroscopy. Soil Science 167, 739–749. Montoneri, E., Savarino, P., Adani, F., Genevini, P.L., Ricca, G., Zanetti, F., Paoletti, S., 2003. Polyalkylphenyl-sulphonic acid with acid groups of variable strength from compost. Waste Management 23, 523–535. Mustin, M., 1987. Le Compost. Gestion de la Matie`re Organique. ´ ditions Franc¸ois Dubusc, Paris. E Oleszczuk, P., Baran, S., 2005. Polycyclic aromatic hydrocarbons content in shoots and leaves of willow (Salix viminalis) cultivated on the sewage sludge-amended soil. Water, Air and Soil Pollution 168, 91–111. Oleszczuk, P., in press. Phytotoxicity of municipal sewage sludge composts related to physico-chemical properties, PAHs and heavy metals. Ecotoxicology and Environmental Safety doi:10.1016/j.ecoenv.2007.04.006. Peuravuori, J., Pihlaja, K., 2002. Molecular size distribution and spectroscopic properties of aquatic humic substances. Analytica Chimica Acta 337, 133–149.
2023
Peuravuori, J., 2005. NMR Spectroscopy study of freshwater humic material in light of supramolecular assembly. Environmental Science and Technology 39, 5541–5549. Peuravuori, J., Zˇba´nkova´, P., Pihlaja, K., 2006. Aspects of structural features in lignite and lignite humic acids. Fuel Processing Technology 87, 829–839. Pretsch, E., Clerc, T., Seibl, J., Simon, W., 1998. Tablas para la determinacio´n estructural por me´todos espectrosco´picos. Translation of the third German edition. Springer-Verlag, Barcelona. Riffaldi, R., Levi-Minzi, R., Saviozzi, A., 1983. Humic fractions of organic wastes. Agriculture, Ecosystems and Environment 10, 353–359. Sa´nchez-Monedero, M.A., Cegarra, J., Garcı´a, D., Roig, A., 2002. Chemical and structural evolution of humic acids during organic waste composting. Biodegradation 13, 361–371. Shin, H.-S., Monsallier, J.M., Choppin, G.R., 1999. Spectroscopic and chemical characterizations of molecular size fractionated humic acid. Talanta 50, 641–647. Stevenson, F.J., 1994. Humus Chemistry, second ed. Wiley, New York. Sun, R., Tomkinson, J., 2002. Comparative study of lignins isolated by alkali and ultrasound-assisted alkali extractions from wheat straw. Ultrasonics Sonochemistry 9, 85–93. Wilson, M.A., Ellis, A.V., Lee, G.S.H., Rose, H.R., Lu, X., Young, B.R., 1999. Structure of molecular weight fractions of Bayer humic substances. 1. Low-temperature products. Industrial and Engineering Chemistry Research 38, 4663– 4674.
Organic Geochemistry Organic Geochemistry 38 (2007) 2012–2023 www.elsevier.com/locate/orggeochem
The complementary use of 1H NMR, 13C NMR, FTIR and size exclusion chromatography to investigate the principal structural changes associated with composting of organic materials with diverse origin Marta Fuentes a, Roberto Baigorri b, Gustavo Gonza´lez-Gaitano a, Jose´ Ma Garcı´a-Mina a,b,* a
Department of Chemistry and Soil Chemistry, University of Navarra, 31080 Pamplona, Spain b CIPAV-Roullier Group, Inabonos, Polı´gono Arazuri-Orcoyen, 31160 Orcoyen, Spain
Received 5 December 2006; received in revised form 27 July 2007; accepted 23 August 2007 Available 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 mixtures were 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 acids and two reference fulvic acids (1S104H, 1R103H, 1R101F and 1R107F) obtained from the International Humic Substances Society were also characterized using these techniques, in order to compare the features of reference humic and fulvic acids with those of composted materials. Likewise, the results were compared with those obtained in previous studies, in which UV–Visible and fluorescence spectroscopies were employed to characterize the humification degree of the molecular systems. The results obtained by 13C CPMAS NMR, 1H NMR and FTIR indicate that, in general, humification seems to be associated with an increase in the aromatic character of the systems, with the presence of phenol groups as principal substituents and a reduction in oxygen containing functional groups, principally carboxylic or carbonylic groups, as well as the development of molecular fractions with larger size. These results also support the suitability of UV–Visible and fluorescence spectroscopies in the assessment of the humification course of humic extracts in composting processes. Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction *
Corresponding author. Address: Department of Chemistry and Soil Chemistry, University of Navarra, 31080 Pamplona, Spain. Tel.: +34 948324550; fax: +34 948324032. E-mail address: [email protected] (J.M. Garcı´a-Mina).
Recycling organic wastes from industrial and domestic sources by means of composting has arisen in recent years as an alternative to classical soil
0146-6380/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2007.08.007
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
amendment (Gonza´lez-Vila et al., 1999; Ait Baddi et al., 2004a,b; Amir et al., 2004). These organic residues have to be pretreated in order to stabilize them and to reduce their initial toxicity. Non-composted residues or immature composts applied to agricultural soils degrade rapidly, and may cause phytotoxicity due to the presence of simple organic acids, and decreases in oxygen and nitrogen concentrations, among other harmful effects on soil properties (Mustin, 1987; Jime´nez and Garcı´a, 1992). The content in potential organic (i.e., polycyclic aromatic hydrocarbons) and inorganic (heavy metals) pollutants of composts must also be controlled, as these contaminants may migrate to groundwater or accumulate in plants (Oleszczuk and Baran, 2005; Alvarenga et al., 2007; Oleszczuk, in press). The quality and stability of composts depend largely on the properties of the initial materials, the time of composting and the oxidative conditions of treatment that influence microbial activities, resulting in different degrees of degradation and transformation of the initial organic mixtures. Composting has been described as an accelerated version of the decomposition processes naturally occurring in the soil (Ait Baddi et al., 2004a). Therefore, the characterization of the changes in the composition of the compost and its comparison with reference humic substances generated in soils are two important points to consider when assessing the quality and properties of the final products, and studying the singular structural changes and features associated with humification. These changes have classically been attributed to increases in aromatic content in humic substances, although other studies have shown that accumulation of recalcitrant aliphatic structures also takes place during composting (Almendros et al., 2000). Organic matter transformation during composting can be estimated by C/N ratios, soluble organic carbon concentration in water extracts or humic acid to fulvic acid ratio (Hsu and Lo, 1999; Jouraiphy et al., 2005). Indices derived from fluorescence and UV–Visible spectroscopies have also proven to be useful in evaluating the humification degree of HS extracted from organic materials of diverse origin (Korshin et al., 1997; Peuravuori and Pihlaja, 2002; Milori et al., 2002; Fuentes et al., 2006), as well as Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopies (NMR) (Gonza´lez-Vila et al., 1999; Sa´nchez-Monedero et al., 2002; Ait Baddi et al., 2004a; Amir et al., 2004; Jouraiphy et al., 2005).
2013
In a previous study (Fuentes et al., 2006), extracts obtained with alkaline solutions from several composted materials and from their initial raw organic mixtures, in addition to various humic and fulvic acids from soils, including International Humic Substances Society (IHSS) reference materials, were studied using UV–Visible and fluorescence spectroscopies. Several indices indicated reasonably well the evolution (humification) of the different systems, indicating that humification might be related to: (i) significant changes in the aromatic character of the samples, which in turn could be associated with more functional ring substitution and polycondensation; and (ii) significant changes in the molecular size distribution throughout humification. In order to study more in depth these possible structural changes associated with humification, we have investigated the same organic systems characterized by UV–Visible and fluorescence spectroscopies (Fuentes et al., 2006) through the complementary use of elemental analysis, 1H NMR, 13C NMR, FTIR and high pressure size exclusion chromatography (HPSEC). Likewise, this study has permitted us to compare the properties of composted materials with those of humic and fulvic acids extracted from soils. 2. Materials and methods 2.1. Organic materials The organic systems studied were divided into three groups: (i) humic substances (humic and fulvic acids) with diverse origin, including IHSS standards; (ii) organic substances contained in alkaline extracts obtained from composted materials; and (iii) organic substances contained in alkaline extracts obtained from the initial organic materials used in the different composting procedures. Soil humic substances were isolated from soil samples of different locations: China (CHHA) and Czech Republic (CZHA). Commercial humic acid was 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 composted and non-composted raw materials were defined 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 initial mill olive residues (OLV); compost of domestic wastes (DWC) and initial domestic wastes (DW); compost of ovine manures plus straw (OVC) and straw (STW); compost of a mixture of animal manures (FMC) and the initial mixture (noncomposted) of animal manures (FM). The characterization of these materials is summarized in Table 1. The starting materials were piled in rows, and these piles were periodically turned over in order to aerate and homogenize the mixtures. The time of composting was four months, and during this period piles were turned six times.
by a LECO CHN 900 analyser. The oxygen content was determined by difference (ash-free basis). 2.4.
13
C NMR spectroscopy
13
C NMR spectra were obtained on a Varian Unity 300 spectrometer at 75.429 MHz using the cross-polarization magic angle spinning technique (CPMAS), with a spinning speed of 5 kHz, 90° pulse width, 69 ms acquisition time and 1.0 s delay. 2.5. 1H NMR spectroscopy Solution 1H NMR spectra were recorded on a Varian Unity-300 spectrometer at 300 MHz, using a 5 mm multinuclear probe, with 90° pulse angle, a sweep width of 4000 Hz and a line broadening of 0.5 Hz. Gated irradiation was applied between acquisitions 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 shift standard.
2.2. Extraction procedure The different organic systems were extracted from solid samples of the organic materials with 0.1 M NaOH (24 h of mechanical shaking in darkness) at 22 °C. The air was displaced by N2 in the extracts in order to avoid possible oxidation during the extraction process. The sample:extractant ratio used was 1:6 for all samples but 1:10 for straw (this needed a larger volume of NaOH solution because of its great liquid absorption capacity). The suspension was then centrifuged at 11,100 g for 15 min and the alkaline supernatants were treated with an acidic-cation exchange resin (Amberlite IR-120H, Aldrich) in order to lower the pH to 3.5 before freeze-drying.
2.6. FTIR spectroscopy Pellets were prepared by mixing 1 mg of each freeze-dried sample with 100 mg of KBr so that the mixture became homogeneous. Infrared spectra were recorded on these pellets with a Nicolet Magna-IR 550 spectrometer over the 4000–400 cm 1 range.
2.3. Elemental analysis
2.7. HPSEC study
The carbon, hydrogen and nitrogen contents of the lyophilized samples were analysed in duplicate
Molecular size distribution for humic materials was evaluated by high performance size exclusion
Table 1 Moisture, ash content and elemental composition (w/w %) of the initial organic mixtures and the corresponding composted materials Organic substances
Moisturea
Ashb
Cc
Hc
Nc
Oc
C/N
C/H
Ovine manure
STW OVC
8.9 8.0
0.6 7.0
48.2 51.3
5.8 5.3
0.3 4.3
45.6 39.1
177.9 14.0
0.69 0.81
Animal manure
FM FMC
8.0 4.6
11.1 10.7
57.4 53.3
6.2 6.2
8.0 7.2
28.4 33.4
8.4 8.7
0.77 0.72
Olive wastes
OLV OLVC
9.1 8.4
2.5 4.0
54.0 56.2
6.5 5.2
1.5 2.6
38.0 36.0
41.3 25.5
0.69 0.90
Grape wastes
GW GWC
9.5 12.0
3.1 5.5
52.4 58.0
5.7 5.1
1.9 3.7
40.0 33.2
32.7 18.3
0.77 0.94
Domestic wastes
DW DWC
7.2 8.4
2.7 7.4
43.2 54.5
4.6 5.6
1.3 4.8
50.9 35.1
38.7 13.3
0.78 0.82
a b c
Wet matter. Dry matter. Dry and ash-free matter.
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
2015
3.1. Elemental analysis
chromatography. The chromatographic system consisted of a Waters 600 Controller pump followed by two detectors in series: a Waters 996 Photodiode Array Detector set at 400 nm, and a Waters 2424 refractive index detector (RI). Size exclusion separation occurred through a PL Aquagel-OH 30 column (Polymer Laboratories), preceded by a guard column with the same stationary phase. The overall molecular weight range of separation for this column is 100–300,000 Da. For each sample, solutions of 800 mg of organic carbon l 1 were prepared in 0.05 M NaNO3. The injection volume of all samples was 100 ll, the eluent used was 0.05 M NaNO3 (pH 7), and the flow rate was 1 ml/min. Void volume (V0 = 6.65 ml) and permeation volume (Vp = 11.82 ml) were determined with polyethylene oxide of MW of 43,250 Da and methanol, respectively.
The HA studied (CHHA, CZHA, AHA, LSHA, PRHA) presented the highest content in carbon, around 55–60% (Table 2). Reference fulvic acids showed slightly lower values, but they had higher oxygen content, mainly due to the larger content in COOH groups in those compounds as shown by 13C NMR (Table 3). Values for O/C atomic ratios fell within the range described in the literature for HA and FA. Thus, HAs presented O/C values around 0.5, whereas those of FAs were close to 0.7 (Stevenson, 1994). Differences in the nitrogen content were not significant, although C/N ratio varied, being lower in HAs and indicating larger nitrogen fixation in these substances. As for the organic substances extracted from composted and non-composted materials, the carbon content decreased in OVC, OLVC and FMC, but increased in GWC and DWC. The percentage of H and C/N ratios decreased in all cases. The C/H ratios increased in all cases to varying degrees, while the N content increased in all cases except FMC. The increase in the C/H ratio suggests that changes in chemical composition during the composting
3. Results and discussion The results obtained using the different techniques will be presented and commented upon separately, and afterwards these results will be related to those previously obtained using UV–Vis and fluorescence spectroscopies as described in Fuentes et al. (2006).
Table 2 Elemental analysis for HAs and FAs from soils and IHSS and for humic-like substances extracted from raw initial organic mixtures and composts (see acronyms in the text, Section 2.1) Families
Organic substances
Elemental analysis %C
%H
%N
%O
O/C
HS
CHHA CZHA AHA
59.6 59.4 53.4
1.6 2.4 3.7
1.4 1.3 0.7
37.4 36.8 42.2
0.47 0.46 0.59
49.7 53.3 89.0
3.10 2.06 1.20
HS-IHSS
LSHA PRHA SRFA WRFA
62.2 55.7 52.5 53.5
3.6 3.8 4.3 4.2
1.2 3.6 0.7 1.1
30.5 36.9 43.5 41.7
0.37 0.50 0.62 0.58
60.5 18.0 87.5 56.7
1.44 1.22 1.02 1.06
Ovine manure
STW OVC FM FMC
40.1 33.7 43.2 34.2
3.7 3.0 4.4 3.7
0.6 3.7 10.0 7.7
55.5 59.5 42.4 54.5
1.04 1.32 0.74 1.19
78.0 10.6 5.0 5.2
0.90 0.94 0.82 0.78
Olive wastes
OLV OLVC
56.0 48.7
5.0 4.1
0.6 2.5
38.4 44.7
0.51 0.69
108.9 22.7
0.93 0.99
Grape wastes
GW GWC
38.9 48.1
3.9 3.2
1.0 4.6
56.3 44.1
1.08 0.68
45.4 12.2
0.83 1.25
Domestic wastes
DW DWC
39.1 41.4
5.1 3.3
1.4 7.1
54.4 48.2
1.04 0.78
32.6 6.8
0.63 1.04
Animal manure
Atomic ratios C/N
C/H
2016 Table 3 Relative abundances of different carbon and hydrogen types (in %) measured by 13C CPMAS NMR and 1H NMR spectroscopy, and parameters obtained by UV–Vis and fluorescence spectroscopies Families
Organic substances
13
1 H NMR (Chemical shift range in ppm)
C NMR (Chemical shift range in ppm) 45–110
110–160
140–160
160–215
0.5–3
3–4.5
6–8.5
HarH/CarC
EET =EBz b
e280 c
e600 d
A440e
Alkyl C
O-alkyl C
Aromatic C
Phenolic C
Carbonylic C
Hal
Ha
Har
HS
CHHA CZHA AHA
13.8 27.4 62.5
3.2 12.6 18.8
79.5 46.5 12.1
5.4 12.8 5.3
3.5 13.5 6.6
24 70 42
53 17 16
23 13 42
0.09 0.13 3.03
0.90 0.86 0.84
980 1064 777
92.5 51.9 50.6
10230 16160 8110
HS-IHSS
LSHA PRHA SRFA WRFA
26.6 20.1 32.5 23.2
14.9 10.9 14.2 12.2
48.7 37.3 15.6 29.0
12.8 12.4 5.0 9.8
9.8 31.7 37.7 35.6
31 52 71 37
68 40 22 36
1 8 7 27
0.01 0.18 0.47 0.88
0.82 0.77 0.62 0.67
1402 834 449 500
80.1 58.3 46.5 16.3
8540 5970 4490 4750
Ovine manure
STW OVC
18.6 17.7
49.7 25.0
8.2 22.3
3.3 10.0
23.5 35
26 48
65 46
9 6
1.21 0.26
0.45 0.50
116 373
2.0 14.7
1010 4150
Animal manure
FM FMC
33.7 30.4
13.8 24.0
11.8 9.6
4.0 3.0
40.7 36
72 27
24 68
4 5
0.45 0.68
0.41 0.56
108 286
3.0 9.8
2040 4110
Olive wastes
OLV OLVC
23.2 22.2
56.4 29.3
11.2 19.7
5.4 7.1
9.2 28.8
40 45
58 47
2 8
0.23 0.42
0.47 0.65
106 256
13.2 16.9
1840 4550
Grape wastes
GW GWC
3.5 21.6
46.9 13.1
1.5 26.8
1.0 10.0
48.1 38.5
28 56
70 43
2 1
1.33 0.15
0.33 0.76
55 415
3.1 19.5
780 4480
Domestic wastes
DW DWC
23.7 25.4
28.2 34.9
20.3 16.4
9.5 5.0
27.8 23.3
61 56
36 39
3 5
0.26 0.32
0.51 0.62
70 354
2.8 26.4
680 3830
a b c d e
Values presented for EET/EBz, e280, e600 and A440 are taken from a previous study (Fuentes et al., 2006). Ratio of absorbances at 253 and 220 nm in the UV spectrum. Molar absorptivity at 280 nm (L cm 1 mol 1 of organic carbon). Molar absorptivity at 600 nm (L cm 1 mol 1 of organic carbon). Area under fluorescence emission spectra (460–650 nm) with kexc = 440 nm.
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
0–45
UV–Vis and fluorescence parametersa
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
process could be due to the degradation of substances with low C/H, such as carbohydrates, polysaccharides or fatty acids, along with an increase in unsaturated structures relative to saturated ones (Miiki et al., 1997; Sa´nchez-Monedero et al., 2002). Nitrogen compounds are thought to be incorporated to the humic ‘‘core’’ through condensation of proteins and modified lignin, reactions between N-containing compounds and quinones derived from lignin, or sugar-amine condensation (Stevenson, 1994). The absence of significant differences in the chemical composition corresponding to the organic substances extracted from fresh and composted materials has also been found by other authors (Riffaldi et al., 1983; Inbar et al., 1990; Ait Baddi et al., 2004a), who reported that the chemical composition of humic-like substances extracted from various organic sources at different stages of maturity remained almost unchanged during composting. On the other hand, the organic substances extracted from composted materials presented C/H ratio values similar to those of fulvic acids and lower than those of humic acids. These results could indicate the presence of less aromatic structures in the organic substances extracted from composted materials compared to the soil humic acids. Finally, the high nitrogen values found in the organic substances extracted from composted materials could be indicative of high contents of non-humified biomolecules (such as polysaccharides and polypeptides) or to the incomplete hydrolysis of proteinaceous constituents in these systems (Sa´nchez-Monedero et al., 2002; Ait Baddi et al., 2004a). 3.2.
13
C NMR study
In general the main trends inferred from the elemental analysis study were confirmed by the results obtained in the 13C NMR study. The 13C NMR spectra are shown in Fig. 1. For data analysis, spectra were divided into chemical shift regions assigned to the following classes of chemical 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 carbonyl C (160–220 ppm). The relative intensity of these regions was determined by the integration of the corresponding peak areas (Malcolm, 1989; Stevenson, 1994; Gonda et al., 2005).
2017
Significant changes were observed between GWC, OLVC, OVC and their respective initial fresh organic materials GW, OLV, STW. In the three cases the aliphatic content decreased due to a loss of alkyl carbon bonded to oxygen, whereas percentages of aromatic, phenolic, and carbonylic C increased (Table 3). These changes may reflect that during composting, the unstable organic compounds such as aliphatic materials are transformed through intense microbial activities into more stable humic compounds with more oxidized, olefinic or aromatic structures that could include more polycondensed rings (Amir et al., 2004). In the case of DW–DWC and FM–FMC systems, however, the distribution of functional groups did not undergo important variations, which mainly consisted of an increase in aliphatic C (mainly O-alkyl C) and a slight decrease in aromatic and carbonylic C (Table 3). Gonza´lez-Vila et al. (1999) studied composts from urban wastes and did not distinguish a clear progressive trend of the organic components in the course of the composting process. Composting led to simultaneous degradation of all types of C, with no selective accumulation of any preferentially stable forms. Almendros et al. (2000) found that in most cases for composted forest and shrub biomass, the recalcitrant material accumulated during composting was not exclusively aromatic in nature and that the presence of tannins may contribute, through their selective preservation and condensation reactions, to limiting the decomposition of aliphatic biomacromolecules. Likewise, these authors indicated that the decrease in the carboxylic content might be due to the degradation of lipids. These authors also emphasize the possible interfering signals of protein moieties in the humic substances extracted from composts, as amino acids contribute to resonance in the alkyl C, Oalkyl C and carbonyl C regions. On the other hand, soil humic acids showed lower aliphatic content (mainly methyl and methylene groups) and higher aromatic content when compared with the organic substances extracted from composted materials, except in the case of the Aldrich humic acid (AHA) that was mostly aliphatic, in agreement with what had been previously reported (Malcolm and MacCarthy, 1986; Shin et al., 1999). It was noteworthy the similarities in functional groups between the organic substances extracted from composted materials and the IHSS fulvic acids.
2018
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
a
b STW
CHHA OVC FM
CZHA
FMC AHA OLV LSHA
OLVC
PRHA
GW GWC
SRFA DW WRFA
DWC 225 200 175 150 125 100 75
50
25
0
225 200 175 150 125 100 75
50
25
0
δ (ppm)
δ (ppm)
Fig. 1. 13C CPMAS NMR spectra of: (a) HAs and FAs from soils and IHSS; and (b) humic extracts from non-composted and composted organic materials.
3.3. 1H NMR study Several representative 1H NMR spectra recorded for the different systems studied are shown in Fig. 2. Proton spectra are typically divided into three main regions: (i) 0.5–3 ppm, resonance of alkyl protons and protons attached to carbon in a to aromatic
PRHA D2O peak
WRFA
OLV
OLVC 8
6
4
2
0
δ (ppm) 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 oxygen or nitrogen (Ha); and (iii) 6–8.5 ppm, protons attached to unsaturated carbons and aromatic protons (Har) (Lambert and Lankes, 2002). The integration of these areas and the percentage of each type of proton are shown in Table 3. In the region of resonance of aliphatic protons, peaks around 0.9 ppm are assigned to protons of terminal methyl groups, whereas signals between 1.2 and 1.8 ppm are attributed to methylene and methine protons. The peaks appearing in the range of 2.0–2.8 ppm can be assigned to protons adjacent to functional groups with electronegative atoms (carboxyl, amide, carbonyl or ester groups). Resonances for proteins also appear between 1.5 and 2.8 ppm. The signals covering the 3.2–4.5 ppm range include contributions from H on Ca to an oxygen or nitrogen atom (Chen et al., 2000; Adani and Ricca, 2004; Kovac et al., 2004). Protons attached to aminomethine and/or methylene groups bonded to amide functional groups show resonance at 3.1–3.35 ppm (Montoneri et al., 2003), although more intense signals appear at 3.7–3.9 ppm, attributed to CHOH and CH2OH groups. In the case of composts, these functional groups may indicate the presence of methoxyphenylpropyl repeating units which typically occur in lignin, and/or the
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
presence of polysaccharide moieties (Adani et al., 2006). In the case of GWC, OLVC and OVC, an increase in Hal and Har and a decrease in Ha content is observed when compared with GW, OLV and STW, respectively. The loss of Ha percentage and the increase in aromatic protons may be related to the corresponding decrease in O-alkyl C and increase in aromatic C observed by 13C NMR. On the other hand, FMC and DWC showed a decrease in aliphatic protons and an increase in Ha and Har, supported by the changes observed by 13C NMR (Table 3). Comparison of proton and carbon aromaticities has been reported to be useful (Lee et al., 1998; Wilson et al., 1999; Peuravuori, 2005; Peuravuori et al., 2006). Thus, high carbon aromaticity and low proton aromaticity would reflect a high degree of aromatic ring substitution or condensation. With this in mind, HarH/CarC ratios were calculated (Table 3, H/C is the hydrogen/carbon ratio). Fulvic acids showed greater values than humic acids, indicating that to some extent humic acids possess more substituents on the aromatic rings and/or more condensed aromatic structures than fulvic acids. The results also showed that humic and fulvic acids presented a higher degree of aromatic carbon substitution and/or condensed aromatic rings when compared with the organic substances extracted from composted and non-composted materials. This ratio did not have a clear trend in composted materials, experiencing a decrease in GWC and OVC but an increase in DWC, FMC and OLVC. 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@CAH resonances, and this is specially evident in the case of AHA, STW and GW, that yielded HarH/CarC ratios higher than 1. 3.4. FTIR spectroscopy The FTIR spectra of the THE from composted materials exhibited the same peaks as those extracted from fresh organic materials, but these spectra differed in the relative intensity of some bands (Fig. 3). The broad band centered at around 3400 cm 1 corresponds to O–H stretching of hydroxyl groups of alcohols, phenols and organic acids, as well as N–H groups; peaks at 2925 and 2850 cm 1 are caused by symmetric and asymmetric stretching vibrations of C–H in CH2 and CH3 groups. The band (a shoulder in some cases) at 1715 cm 1 is
2019
3430
1570 2925
1630 1400
2850
1120 1045
1715
STW
OVC
FM
FMC
OLV
OLVC
GW
GWC
DW
DWC
4000
3500
3000
2500
2000
1500
1000
500
ν (cm-1)
Fig. 3. FTIR spectra of humic extracts from non-composted and composted organic materials.
attributed to the C@O stretching vibration of COOH, ketones, aldehydes and esters. The band centered at around 1640–1620 cm 1 may be related to aromatic C@C stretching and C@O stretching of quinone and/or conjugated ketone and amide groups (amide I). The shoulder appearing at 1460– 1450 cm 1 is generated by aliphatic C–H deformations and aromatic ring vibrations. The peak at 1400–1390 cm 1 is attributed to O–H deformation, C–O stretching of phenolic OH and C–H deformation of CH2 and CH3 groups. A weak band between 1260 and 1220 cm 1 is produced by amides or ethers, and a broad band between 1120 and 980 cm 1 with a sharp peak centered near 1045 cm 1 is related to C–O stretching of polysaccharide or polysaccharide-like substances, as well
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-plain deformation 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 during the different composting processes were reflected by a decrease in the bands at 2925–2850 cm 1 (except for FMC) and at 1040 cm 1, and an increase in those at 1715, 1640, 1460 and 1400 cm 1. The rise in these bands suggests an increase in carbonyl groups (COOH, ketones, aldehydes, esters) as well as aromatic, phenolic and quinone structures. These changes observed by FTIR indicate the decrease, during composting, in aliphatic and polysaccharide structures and the increase in more oxidized and, probably, polycondensed aromatic components. In general, these results are in line with those obtained in the 13C NMR study. Humic and fulvic acids have similar spectra (Fig. 4), the main difference being that the intensity of the 1715 cm 1 band is considerably stronger in fulvic acids because of the occurrence of more COOH groups. As shown by 13C NMR spectra, fulvic acids have greater content in carbonyl C than in aromatic C.
3430
In this study, no attempt was made to determine the absolute molecular weight of humic substances. Chromatograms were only used for relative comparison of molecular size distributions between the different systems. As can be seen in Fig. 5, the chromatograms presented different profiles between humic or fulvic acids extracted from soils, THE from composted materials and those from fresh organic raw mixture. However, samples belonging to the same group showed a similar pattern. THE from composted materials showed an elution profile corresponding to higher molecular sizes than those corresponding to THE from non-composted materials. Several authors (Sa´nchez-Monedero et al., 2002) have also observed an increase in the average molecular weight of humic substances extracted from different composts, suggesting that this could be the result of condensation reactions of different fractions to the humic ‘‘core’’ producing macromolecules, or to the degradation of the smallest fractions and the consequent enrichment of the
1620 2850
1240
1715 1400
1040
CHHA
CZHA
AHA
LSHA
PRHA
SRFA
WRFA
4000
3.5. HPSEC
2925
3500
3000
2500
2000 ν (cm-1)
1500
1000
500
Fig. 4. FTIR spectra of HAs and FAs from soils and IHSS.
largest ones. Other authors attribute this to biodegradation followed by the formation of more polycondensed 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 parameters derived from the UV–Visible and fluorescence studies (Fuentes et al., 2006) (Table 3) and the structural features derived from this study (Table 3), different general patterns were observed depending on the organic systems included in the comparison. Thus, when all organic systems (soil humic acids, IHSS standards, THE from composted and from fresh materials) are considered, a significant positive correlation (P < 0.05, data not shown) was observed between UV–Visible and fluorescence indexes (e280, e600, EET/EBz, A440 and A4/A1, defined in Table 3
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023
LSHA
WRFA
DW
DWC
OLV
OLVC 0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
Ve (mL) Fig. 5. HPSEC chromatograms (recorded with the refractive index detector) of a humic acid (LSHA), a fulvic acid (WRFA), and the humic extracts from two raw initial mixtures (DW and OLV) and their corresponding composted material (DWC and OLVC).
footnote) and % total C, C/H ratio, and the percentages of aromatic C and phenolic C. These results were also associated with significant negative correlations between the UV–Visible and fluorescence indexes and % H, % O, O/C ratio and, in some cases, with carboxylic C. These results seem to indicate that humification is associated with both more aromatic rings in the structure with the presence of phenol groups, and less oxygen containing functional groups, principally carboxylic groups. Regarding the possibility that humification is also related with more condensation in the aromatic moieties, our results do not permit us to assess this question. In fact no significant correlations between the HarH/CarC ratio and the different UV–Visible and fluorescence parameters were observed. However, when only THE from composted and fresh materials are considered, in general the same patterns (the main difference consisted of an increase in the carboxylic C with composting) are observed, except in the case of FM–FMC and DW–DWC systems (Table 3). In these systems,
2021
the general increase in the values of UV–Visible and fluorescence indices with composting was not correlated with increases in the aromatic character or the presence of phenol and carboxylic groups. This result indicates that other structural features in addition to those related to the total aromatic character must be related to the increase in UV–Visible and fluorescence indices. This structural property could be related to the condensation degree in aromatic rings and/or the conjugation degree in aromatic and aliphatic moieties. In summary, these results seem to indicate two different phases associated with humification. A first phase consisting of the first transformation of fresh plant and microbial material into more humified materials, that could be represented in this study by THE from fresh and composted materials, in which the humification associated with composting is related to increases in the aromatic character and the presence of more acidic functional groups in the structure. This process seems to be also associated with the development of molecular fractions with larger sizes. THE from composted materials presented some structural features and values of the UV–Visible and fluorescence indexes similar to those of IHSS reference fulvic acids. However, the results obtained also indicate that more advanced stages of humification involve increases in both the aromatic character that could be linked to a higher degree of polycondensation, and the presence of phenol groups; and a decrease in carboxylic groups that might be related to aromatic condensation processes producing aromatic quinone-type structures. 4. Conclusions Humic extracts from composted materials yielded higher C/H and lower C/N ratios than humic extracts from their initial fresh organic mixtures. These changes could be the result of the degradation of carbohydrates, polysaccharides or fatty acids, and the incorporation into the humic ‘‘core’’ of N containing groups through condensation of proteins and modified lignin, or sugar-amine condensation. FTIR and 13C NMR spectroscopies showed a decrease in aliphatic content and an increment in polar functional groups (O- or N-containing functional groups) as a result of composting. When compared with FA and HA, THE from composted materials showed more structural similarities with 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 the degree of evolution (humification) of the composted materials. e280 reflects the aromatic content, while EET/EBz ratio seems to be an indicator of the degree of substitution in the aromatic ring with polar functional groups, as the results from FTIR and 13 C NMR suggest. Likewise, A440 parameter may be related to the degree of complexity in the aromatic moiety. Finally, these results also indicate that humification seems to be associated with both an increase in the aromatic character of the system with the presence of phenol groups as principal substituents, and a reduction in oxygen containing functional groups principally carboxylic groups. Likewise some of these results indicate that increases in aromatic condensation and the degree of conjugation could be also involved during humification. More studies, therefore, are needed in order to elucidate these questions. In particular, specific 13C NMR and pyrolysis-MS studies could prove very useful. Acknowledgements This research was founded by the Roullier Group and the Government of Navarra. Special thanks to David Rhymes for kindly improving the English of the manuscript. Associate Editor—Ian D. Bull References Adani, F., Ricca, G., 2004. The contribution of alkali soluble (humic acid-like) and unhydrolyzed-alkali soluble (corehumic acid-like) fractions extracted from maize plant to the formation of soil humic acid. Chemosphere 56, 13–22. Adani, F., Genevini, P., Tambone, F., Montoneri, E., 2006. Compost effect on soil humic acid: a NMR study. Chemosphere 65, 1300–1307. Ait Baddi, G., Hafidi, M., Cegarra, J., Alburquerque, J.A., Gonza´lvez, J., Gilard, V., Revel, J.-C., 2004a. Characterization of fulvic acids by elemental and spectroscopic (FTIR and 13 C NMR) analyses during composting of olive mill wastes plus straw. Bioresource Technology 93, 285–290. Ait Baddi, G., Alburquerque, J.A., Gonza´lvez, J., Cegarra, J., Hafidi, M., 2004b. Chemical and spectroscopic analyses of organic matter transformations during composting of olive mill wastes. International Biodeterioration and Biodegradation 54, 39–44. Almendros, G., Dorado, J., Gonza´lez-Vila, F.J., Blanco, M.J., Lankes, U., 2000. 13C NMR assessment of decomposition patterns during composting of forest and shrub biomass. Soil Biology and Biochemistry 32, 793–804.
Amir, S., Hafidi, M., Merlina, G., Hamdi, H., Revel, J.-C., 2004. Elemental analysis, FTIR and 13C NMR of humic acids from sewage sludge composting. Agronomie 24, 13–18. Alvarenga, P., Palma, P., Gonc¸alves, A.P., Fernandes, R.M., Cunha-Queda, A.C., Duarte, E., Vallini, G., 2007. Evaluation of chemical and ecotoxicological characteristics of biodegradable organic residues for application to agricultural land. Environment International 33, 505–513. Chen, Y., Katan, J., Gamliel, A., Aviad, T., Snitzer, M., 2000. Involvement of soluble organic matter in increased plant growth in solarized soils. Biology and Fertility of Soils 32, 28– 34. Fuentes, M., Gonza´lez-Gaitano, G., Garcı´a-Mina, J.M., 2006. The usefulness of UV–visible and fluorescence spectroscopies to study the chemical nature of humic substances from soils and composts. Organic Geochemistry 37, 1949– 1959. Gonda, D., Lopez, R., Fiol, S., Antelo, J.M., Arce, F., 2005. Characterization and acid–base properties of fulvic and humic acids isolated from two horizons of an ombrotrophic peat bog. Geoderma 126, 367–374. Gonza´lez-Vila, F.J., Almendros, G., Madrid, F., 1999. Molecular alterations of organic fractions from urban waste in the course of composting and their further transformation in amended soil. The Science of the Total Environment 236, 215–229. Hsu, J.-H., Lo, S.-L., 1999. Chemical and spectroscopic analyses of organic matter transformations during composting of pig manure. Environmental Pollution 104, 189–196. Inbar, Y., Chen, Y., Hadar, Y., 1990. Humic substances formed during the composting of organic matter. Soil Science Society of America Journal 54, 1316–1323. Jime´nez, E.I., Garcı´a, V.P., 1992. Determination of maturity indices for city refuse composts. Agriculture Ecosystems and Environment 38, 331–343. Jouraiphy, A., Amir, S., El Gharous, M., Revel, J.C., Hafidi, M., 2005. Chemical and spectroscopic analysis of organic matter transformation during composting of sewage sludge and green plant waste. International Biodeterioration and Biodegradation 56, 101–108. Korshin, G.V., Li, C.W., Benjamin, M.M., 1997. Monitoring the properties of natural organic matter through UV spectroscopy: a consistent theory. Water Research 31, 1787–1795. Kovac, N., Faganeli, J., Bajt, O., Sket, B., Orel, B., Penna, N., 2004. Chemical composition of macroaggregates in the northern Adriatic Sea. Organic Geochemistry 35, 1095– 1104. Lambert, J., Lankes, U., 2002. Application of Nuclear Magnetic Resonance spectroscopy to structural investigations of refractory organic substances – principles and definitions. In: Frimmel, F.H., Abbt-Braun, G., Heumann, K.G., Hock, B., Lu¨demann, H.-D., Spiteller, M. (Eds.), Refractory Organic Substances in the Environment. Wiley-VCH, Weinheim, pp. 89–95. Lee, G.S.H., Wilson, M.A., Young, B.R., 1998. The application of the ‘‘WATERGATE’’ suppression technique for analyzing humic substances by nuclear magnetic resonance. Organic Geochemistry 28, 549–559. Malcolm, R.L., MacCarthy, P., 1986. Limitations in the use of commercial humic acids in water and soil research. Environmental Science and Technology 20, 904–911.
M. Fuentes et al. / Organic Geochemistry 38 (2007) 2012–2023 Malcolm, R.L., 1989. Applications of solid-state 13C NMR spectroscopy to geochemical studies of humic substances. In: Hayes, M.H.B., MacCarthy, P., Malcolm, R.L., Swift, R.S. (Eds.), Humic Substances II: In Search of Structure. Wiley, Chichester, pp. 339–372. Miiki, V., Senesi, N., Ha¨nninen, K., 1997. Characterization of humic material formed by composting of domestic and industrial biowastes. Chemosphere 34, 1639–1651. Milori, D.M.B.P., Martin-Neto, L., Bayer, C., Mielniczuk, J., Bagnato, V.S., 2002. Humification degree of soil humic acids determined by fluorescence spectroscopy. Soil Science 167, 739–749. Montoneri, E., Savarino, P., Adani, F., Genevini, P.L., Ricca, G., Zanetti, F., Paoletti, S., 2003. Polyalkylphenyl-sulphonic acid with acid groups of variable strength from compost. Waste Management 23, 523–535. Mustin, M., 1987. Le Compost. Gestion de la Matie`re Organique. ´ ditions Franc¸ois Dubusc, Paris. E Oleszczuk, P., Baran, S., 2005. Polycyclic aromatic hydrocarbons content in shoots and leaves of willow (Salix viminalis) cultivated on the sewage sludge-amended soil. Water, Air and Soil Pollution 168, 91–111. Oleszczuk, P., in press. Phytotoxicity of municipal sewage sludge composts related to physico-chemical properties, PAHs and heavy metals. Ecotoxicology and Environmental Safety doi:10.1016/j.ecoenv.2007.04.006. Peuravuori, J., Pihlaja, K., 2002. Molecular size distribution and spectroscopic properties of aquatic humic substances. Analytica Chimica Acta 337, 133–149.
2023
Peuravuori, J., 2005. NMR Spectroscopy study of freshwater humic material in light of supramolecular assembly. Environmental Science and Technology 39, 5541–5549. Peuravuori, J., Zˇba´nkova´, P., Pihlaja, K., 2006. Aspects of structural features in lignite and lignite humic acids. Fuel Processing Technology 87, 829–839. Pretsch, E., Clerc, T., Seibl, J., Simon, W., 1998. Tablas para la determinacio´n estructural por me´todos espectrosco´picos. Translation of the third German edition. Springer-Verlag, Barcelona. Riffaldi, R., Levi-Minzi, R., Saviozzi, A., 1983. Humic fractions of organic wastes. Agriculture, Ecosystems and Environment 10, 353–359. Sa´nchez-Monedero, M.A., Cegarra, J., Garcı´a, D., Roig, A., 2002. Chemical and structural evolution of humic acids during organic waste composting. Biodegradation 13, 361–371. Shin, H.-S., Monsallier, J.M., Choppin, G.R., 1999. Spectroscopic and chemical characterizations of molecular size fractionated humic acid. Talanta 50, 641–647. Stevenson, F.J., 1994. Humus Chemistry, second ed. Wiley, New York. Sun, R., Tomkinson, J., 2002. Comparative study of lignins isolated by alkali and ultrasound-assisted alkali extractions from wheat straw. Ultrasonics Sonochemistry 9, 85–93. Wilson, M.A., Ellis, A.V., Lee, G.S.H., Rose, H.R., Lu, X., Young, B.R., 1999. Structure of molecular weight fractions of Bayer humic substances. 1. Low-temperature products. Industrial and Engineering Chemistry Research 38, 4663– 4674.