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Elemental and spectroscopic characterization of humic-acid-like compounds during composting of olive mill by-products
Journal of Hazardous Materials 163 (2009) 1289–1297
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Elemental and spectroscopic characterization of humic-acid-like compounds during composting of olive mill by-products Zainab Droussi a , Valeria D’Orazio b , Mohamed Hafidi c , Aaziz Ouatmane a,∗ a
Equipe Environnement et Valorisation des Agro Ressources, BP: 523, FST Beni Mellal, Morocco Dipartimento di Biologia e Chimica Agroforestale e Ambientale, Via Amendola 165/A, 70126 Bari, Italy c Laboratoire d’Ecologie et Environnement, Faculté des Sciences Semlalia, Marrakech, Morocco b
a r t i c l e
i n f o
Article history: Received 22 April 2008 Received in revised form 21 July 2008 Accepted 22 July 2008 Available online 6 August 2008 Keywords: Solid olive mill residues Composting Humic-acid-like Elemental analysis Spectroscopic techniques (Fluorescence FT IR)
a b s t r a c t Humic acids (HAs) were isolated at different stages of composting from two piles of solid olive mill residues (SOR) treated for the first 30 days with tap water (pile C1) or olive mill wastewater (pile C2), for a total composting period of 9 months. The HA fractions were characterized by elemental analysis, UV–visible, Fourier transform infrared and fluorescence spectroscopy in order to monitor humification process and the maturity of the composts. As composting proceeded, the elemental composition of the humic acids showed a decrease in C and H content, and in the C/N ratio, and an increase in N and O contents and in the C/H and O/C ratios. These changes could be attributed to a loss of aliphatic groups and to an increase of aromatic character, polycondensation and degree of oxidation of the HAs. Spectroscopic data agree and support these results, suggesting that the chemical and structural features of the HAs of both composts tend to reach those typical of native soil HAs, that is compounds with a high degree of humification and a high molecular weight and complexity. Therefore, both composting processes seem suitable to produce well-humified organic matter, with important benefits for their use in soil amendment. No differences appeared between the two treatments concerning the humic character of the two final composts. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In most Mediterranean countries, the main processes used for olive oil extraction are still based on the three-phase system which produces enormous quantities of two polluting by-products: olive mill waste water (OMW) and solid olive residues (SOR). These wastes are rich in refractory organic compounds such as phenols, tannins, alcohols, pectin and lipids, which make them very resistant and inappropriate to treatment by conventional biological methods [1]. In general, the most common disposal method for OMW has been limited to evaporation in storage ponds because of the low investment required and the favourable climatic conditions in Mediterranean countries [2]. Nowadays, the composting and/or the co-composting of OMW with SOR represents a successful and suitable method to enhance the quality of organic matter occurring in these materials, with the final production of stable organic compounds that look like native soil organic matter, and particularly humic substances (HS) [3]. Successful application of OMW derived compost both as plant growth medium and plant fertilizer were also
∗ Corresponding author. Tel.: +212 23 48 51 12; fax: +212 23 48 52 01. E-mail address: [email protected] (A. Ouatmane). 0304-3894/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2008.07.136
reported in several recent works [4–6]. Obviously, the evolution of organic matter stabilization during composting is of primordial importance in controlling the efficiency of the process. With this purpose, several physical, chemical and biological techniques and parameters have been proposed [7], and recently compost maturity has been investigated by monitoring the humification degree of these materials through chemical and physico-chemical analyses. Sequi and Benedetti [8] proposed the use of the humification parameters (index, rate and degree of humification) and Tomati et al. [9] suggested monitoring the molecular weight of the humic substances as an index of stability. Advanced physico-chemical techniques such as Fourier transform infrared (FT IR), Fluorescence, and Nuclear Magnetic Resonance (13 C NMR) spectroscopies and thermal analysis, such as Differential Scanning Calorimetry (DSC), have also been used to characterize organic matter of composts and humic substances originating from olive mill wastes [10–12], showing that the composting technique represents a suitable treatment for promoting organic matter humification in these materials, thus improving their agronomic value and minimizing possible negative environmental impact. In recent work good correlations were also observed between common humification indices including the E4/E6 absorption ratio of humic substances and the agronomic value of composts from olive by-products [13]. However, although a wide literature is available on the composting of OMW, relatively
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little information is available on the composting of SOR, and, particularly, on the evolution of humic substances during the composting of these materials. The aim of the present study was to follow the evolution occurring in the humic acid-like fraction during the composting of SOR and their co-composting with OMW by means of chemical and spectroscopic analyses.
Fisher’s least significant difference (LSD) protected t-test. The confidence intervals used were 95, 99, and 99.9%. The symbols used to indicate the P values are † significant at P ≤ 0.05, †† significant at P ≤ 0.01, and ††† significant at P ≤ 0.001. Furthermore, Student’s t-test was applied to evaluate the differences between the HA fractions isolated at the same composting time in the different piles. In this case the symbols used to indicate the P values are *significant at P ≤ 0.05, **significant at P ≤ 0.01, and ***significant at P ≤ 0.001.
2. Materials and methods 2.5. E4 /E6 ratio 2.1. Composting process For both the composting and the co-composting trial, a pile was prepared using 1.5 tonnes of olive oil extraction residues obtained from a three-phase system olive mill in Morocco. During the first 30 days the composting pile (Pile C1) was moistened with tap water while the co-composting pile (Pile C2) was moistened with a total of 0.25 tonnes of olive oil wastewater. The composting process was performed for 267 days during which the piles were forked over periodically and their humidity was maintained at around 50–60%. Biological activity was monitored by measuring the temperature of the piles at a depth of 30 cm. To follow the evolution of the humicacid-like fraction, composite samples were collected at different points from the top to the bottom of the plies after: 3 days (samples 1C1 and 1C2), 31 days (samples 3C1 and 3C2), 61 days (samples 6C1 and 6C2), 195 days (samples 10C1 and 10C2) and 267 days (samples 13C1 and 13 C2). The composite samples were homogenized and immediately stored at −18 ◦ C until analysis. 2.2. Humic acids The HAs were isolated on the basis of their solubility in water as a function of pH, with some modification with respect to the procedure of the International Humic Substances Society (IHSS) [14]. Briefly, to 30 g of each compost sample, previously air-dried and crushed, was added 100 mL 0.1 M KOH. The mixtures were shaken mechanically for 2 h at room temperature (RT, 293 + 2 K), then centrifuged at 5000 rpm for 25 min. This operation was repeated until the supernatant obtained was clear. The combined alkaline supernatants were acidified with 6N H2 SO4 (pH 2), allowed to stand for 24 h at 4 ◦ C, to permit coagulation of the HA fractions, then centrifuged again at 5000 rpm for 25 min. The HA precipitates were purified by dissolution in a minimal volume of 0.1 M KOH, transferred to a dialysis membrane (SpectraPore membrane, MWCO 1000 Daltons) and dialyzed against double distilled water in order to eliminate excess salts. Finally, HAs were freeze-dried for 72 h. Moisture and ash contents were measured by heating the HAs overnight at 105 ◦ C and for 2 h at 550 ◦ C, respectively. 2.3. Elemental analysis (C, H, N, S) Carbon, hydrogen, nitrogen, and sulphur concentrations were determined using a combustion-gas chromatography technique (Fisons EA 1108 Elemental Analyzer, Milan, Italy). The oxygen content was calculated by difference: O% = 100 − (C + H + N + S)%. The instrument was calibrated using a BBOT [2,5-bis-(5-tert-butylbenzoxazol-2-yl)-thiophen] standard (ThermoQuest Italia s.p.a.). The data obtained were corrected for moisture and ash contents. All HA samples were analyzed in triplicate. 2.4. Statistical analysis The elemental composition of HA fractions isolated during the composting time from each pile were statistically analyzed using one-way analysis of variance (ANOVA, F-distribution), followed by
The E4 /E6 ratio was calculated as the ratio of absorbance at 465 and 665 nm measured by a PerkinElmer model Lambda 15 UV–vis spectrophotometer on solutions of 3.0 mg of each HA dissolved in 10 mL of 0.05 M NaHCO3 , with the pH adjusted to 8.3 with NaOH. The E4 /E6 ratio is very useful a spectroscopic index to evaluate the different sources of humic material [15]. In particular, the E4 /E6 ratio is inversely related to molecular weight, C content, aromaticity and polycondensation degree of the organic molecules, whereas a positive correlation has been found between this ratio and O and carboxylic group contents and aliphaticity [15]. 2.6. Fourier transform InfraRed (FT IR) spectroscopy The FT IR spectra were recorded on pellets obtained by pressing under reduced pressure a mixture of 1 mg of HA and 400 mg of dried spectrometry grade KBr using a Nicolet Nexus FT IR spectrophotometer equipped with Nicolet Omnic 6.0 software. Spectra were recorded in the range 4000–400 cm−1 with 2 cm−1 resolution, and 64 scans were performed on each acquisition. 2.7. Fluorescence spectroscopy Fluorescence spectra were recorded on aqueous solutions of 100 mg L−1 HA after overnight equilibration at room temperature, and adjustment to pH 8.0 with 0.05 mol L−1 NaOH, using a Hitachi model F-4500 luminescence spectrophotometer. One-dimensional emission spectra were recorded over the range 380–550 nm at a constant excitation wavelength of 360 nm. Excitation spectra were recorded over the range 300–500 nm at a fixed emission wavelength of 520 nm. Synchronous-scan excitation spectra were measured by simultaneously scanning both the excitation and the emission wavelengths (from 300 to 550 nm), while maintaining a constant, optimized wavelength difference = em − ex = 18 nm. The Total Luminescence (TL) spectra were obtained in the form of excitation/emission matrix (EEM, or contour maps) by scanning the wavelength emission over the range 300–600 nm, while the excitation wavelength was increased sequentially in 5 nm steps from 250 to 500 nm. The EEM spectra were generated from TL spectral data by using Noesys 2.4 software. The fluorescence intensity (FI) values (in arbitrary units) were normalized using a quinine sulphate standard (10 mol in 0.1N H2 SO4 ). 3. Results 3.1. Temperature evolution Temperature evolution during the composting process corresponds to a typical curve of an optimal aerobic composting system [16,17]. In both piles, the temperature increased in a few days from 25 to 60 ◦ C (Fig. 1), and oscillated thereafter between 55 and 65 ◦ C for approximately 2 months. This confirms the high biodegradability of SOR and their ability to produce sufficient heat during composting to ensure the hygienisation of the compost. A similar temperature evolution was reported during composting of OMW
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Fig. 1. Temperature evolution during composting of pile C1 (SOR moistened with tap water) and pile C2 (SOR moistened with OMW).
by Ait Badi et al. [10] and Hachicha et al. [5]. The end of the thermophilic phase was reached after approximately 9 months for the two piles. At this stage both composts could be considered stable. Comparison between the two piles showed that primary addition of OMW to SOR did not affect temperature evolution during the composting experiment. Similar results were reported by Hachicha et al. [5] for a mixture of exhausted olive cake and poultry manure compost that was moistened with OMW during the first 2 months of composting. In contrast, the negative effects on microbial activity of adding OMW, and hence the effects on temperature increases during organic waste composting, have been observed by others authors [18]. The results obtained in the present work could be attributed to the low amounts of OMW used. 3.2. Elemental analysis The elemental compositions of the HAs isolated from the two composts at different stages of the composting process are shown in Table 1. Data reported indicate, for both series, a decrease in C content as composting progresses, but some differences between the two piles are evident with regard to the sampling interval examined. In particular, careful analysis of the composition of HA from series C1 indicates that, with respect to the HA 1C1, the HA 3C1 samples show a significantly lower (P < 0.001) C content, whereas no difference was found in the next sample HA 6C1, as if to suggest a slowdown of the process. The decrease was significantly lower (P < 0.001) in sample HA 10C1 and finally it remained unchanged in HA 13C1. The C content of the HA fractions isolated from the C2 series, initially similar to that of the other series, did not show differences between the HA isolated from the first two samples, then decreased during the whole composting process. In particular, the reduction was about the 4% in samples HA 6C2 (P < 0.01), enhanced markedly (by about 10%) in HA 10C2 (P < 0.001), and fell
slightly (about 3%) in HA 13C2 (P < 0.05) (Table 1). The H content shows a similar trend (Table 1). The N content exhibited the opposite tendency, that is it increased during the whole composting process in the HA fractions of both series, showing a value significantly higher (P < 0.001) in each HA fraction with respect to that of the corresponding previous one. The only exception was sample HA 13C1, characterized by a significant increase at the lower confidence interval (P < 0.01). Similarly, the O content results were significantly higher (P < 0.01) in the HA 3C1 with respect to HA 1C1, unchanged in the next sample HA 6C1, and again increased in the sample HA 10C1. Finally, a slight decrease (P < 0.05) was observed in the HA 13C1 fraction (Table 1). In contrast, the O content of the HA fractions from the C2 series remained unchanged until the third sampling, when it increased significantly (P < 0.001) in HA 10C2, and decreased slightly (P < 0.05) in HA 13C2. Furthermore, with the exception of the first sample, the O content results were always greater, at various confidence intervals, in the HAs from the C1 series with respect to the corresponding HAs from the C2 series. Finally, the S content increased significantly (P < 0.001) in the HA fractions of the C1 series, whereas in the C2 series it was significantly enhanced (P < 0.001) only in the HA fractions isolated from samples 10C2 and 13C2. To better evaluate the evolution of the HA fractions during the composting process as a function of the two different treatments, Student’s t-test was applied to analyze the elemental composition of the corresponding HA samples. For all elements, the HA fractions from to 31 days of composting time result those with the greater differences between the two piles (P < 0.001) (Table 1). In particular, HA 3C2 shows higher C and H contents and lower N, S and O contents with respect to the corresponding HA 3C1. Furthermore, the N content of HA 1- and HA 3C2 was significantly lower and HA 6-, HA 10- and HA 13 C2 significantly higher, with respect to the corresponding HA fractions from pile C1 (Table 1). Additional slight differences are also evident at various
Table 1 Elemental composition (moisture and ash free) of humic acids isolated from pile C1 and pile C2 at different stages of composting Composting time (days)
3 31 61 195 267
C%
N%
H%
S%
O%
C1
C2
C1
C2
C1
C2
C1
C2
C1
C2
70.00 64.63††† 63.38 57.14††† 57.81
70.58 70.02*** 67.12††* 60.47†††* 58.47†*
1.05 2.44††† 2.74††† 5.17††† 5.35††
0.99*** 1.30†††*** 3.40†††*** 5.76†††** 5.43†††*
10.29 8.70††† 8.41† 6.32††† 6.15
10.45 10.34*** 9.10†††* 6.23††† 6.34
0.97 2.46††† 1.48††† 2.60††† 4.47†††
0.68** 1.14*** 1.37* 2.78††† 7.04†††*
17.69 21.77††** 23.99* 28.77††* 26.22†***
17.30 17.20 19.01 24.76††† 22.72†
† †† , and ††† (LSD test) refer to significant differences: P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001 respectively; *, **, and *** (t-test) refer to significant differences: P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001 respectively.
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Table 2 Atomic ratios and E4 /E6 ratios of humic acids isolated from pile C1 and pile C2 at different stages of composting Composting time (days)
3 31 61 195 267
C/N
C/H
O/C
E4 /E6
C1
C2
C1
C2
C1
C2
C1
C2
77.48* 30.88*** 27.02*** 12.89*** 12.60
83.26 62.69 23.05 12.26 12.57
0.57 0.62*** 0.63* 0.75** 0.78
0.56 0.56 0.61 0.81 0.77
0.19 0.25*** 0.28* 0.38* 0.34***
0.18 0.18 0.21 0.31 0.29
2.0 2.9 3.4 6.2 5.7
2.2 2.6 3.4 5.8 6.1
*, **, and *** (t-test) refer to differences significant to P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001 respectively.
confidence intervals between the other HA fractions isolated from the two piles during the whole composting period. In general for both series, with increasing composting time, C/N ratios decreased, whereas C/H and O/C ratios increased (Table 2). To evaluate the differences between the atomic ratios of HA fractions in the two piles Student’s t-test was applied and the corresponding P values are reported in Table 2. The HAs from series C1 showed a lower C/N ratio, at various confidence intervals, with respect to the HAs from C2, reaching similar values only in the last sample (Table 2). The C/H ratio was characterized by similar values in the HA fractions of the first and last samples from both series, whereas it was higher in the HA-3C1 and -6C, and lower in HA 10C1 than in the C2 series (Table 2). Finally, the O/C ratios, with the exception of HA samples from the first sample, were always higher in the HAs from series C1 with respect to the corresponding HAs series C2 (P values in Table 2). In general, the extent of these variations followed the trend described for the corresponding elemental composition, at the end
of the composting process reaching similar values for C/N and C/H ratios, whereas a greater O/C ratio was observed in sample HA 13C1 with respect to HA 13C2 (Table 1). As previously reported [2,9,19,20] these values indicate that, with increasing composting time, N-containing compounds probably increased because proteinaceous materials are incorporated into the HA, and, simultaneously both the oxidation and the proportion of unsaturated structures with respect to saturated structures increased (lower C/H ratios). 3.3. E4 /E6 ratio The E4 /E6 ratios of HAs from both series were similar at the beginning of the composting process, then showed a constant increase with composting time, reaching comparable values in the HAs isolated from the last two stages (10C1 and 13C1, 10C2 and 13C2 samples). In particular, for the HA samples from series C1 the E4 /E6 ratio was markedly enhanced in the first 30 days of composting, reaching a maximum value in the sample HA 10C1. A slight decrease was then observed in HA 13 C1 (Table 2). In contrast, the E4 /E6 ratio of HAs from series C2 showed a more gradual increase as composting progressed, reaching a maximum value in sample HA 13C2 (Table 2). A previous study [15] suggested that the E4 /E6 ratio of HAs is correlated positively with the O content and degree of oxidation, and negatively with C content, aromaticity, polycondensation and molecular weight. In agreement with this study, the increase measured correlated positively to O content and negatively to C content. To verify these relationships between spectroscopic properties and elemental composition of HAs, a linear regression was performed by correlating E4 /E6 data with O and C contents (Fig. 2). As expected, in both cases we found the coefficient of determination values (r2 ) very close to 1, espe-
Fig. 2. Linear regression with corresponding r2 values between, respectively, E4 /E6 ratios and O% (upper) and E4 /E6 ratios and C% (lower) of HAs isolated from both series at different stages of composting.
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cially in the linear regression E4 /E6 vs. C content (r2 values slightly higher). 3.4. FT IR spectra The FT IR spectra of a typical native soil HA and HA fractions 1C1, 6C1 and 13C1 and 1C2, 6C2 and 13C2, are shown in Figs. 3 and 4 respectively. The main absorption bands and corresponding assignments are summarized in Table 3. All spectra feature common and distinctive absorption bands, with some differences in their relative intensity. The main characteristics of these spectra are the following: about 3400 cm−1 (OH stretching and, secondarily, N H stretching of various functional groups); about 3006 cm−1 (stretching of aromatic C H); about 2925 and 2853 cm−1 (asymmetric and symmetric C H stretching respectively of CH2 groups); about 1743 cm−1 (C O stretching of aldehydes and ketones); about 1714 cm−1 (C O stretching of COOH and other carbonyl groups); about 1640 cm−1 (aromatic C C skeletal vibrations, C O stretching of quinone and amide groups (amide I band), C O of H-bonded conjugated ketones); about 1540 and 1507 cm−1 (preferentially ascribed to N H deformation and C N stretching of amides (amide II band); about 1460 cm−1 (C H bending of CH3 groups); about 1420 cm−1 (O H deformation and C O stretching of phenolic OH); about 1380 cm−1 (C H deformation of CH2 and CH3 groups, and/or antisymmetric stretching of COO− groups); about 1265 cm−1 (C O stretching of aryl esteres); about 1220 cm−1 (C O stretching of aryl ethers and phenols); about 1120 cm−1 (C O stretching of secondary alcohols and/or ethers); and, finally, about 1040 cm−1 (C O stretching of polysaccharides or polysaccharide-like substances). With increasing composting time, FT IR spectra of HAs from both series show the disappearance of bands at 3006 and 1743 cm−1 , and a decrease in the relative intensity of absorption bands at 2925 and 2853 cm−1 , 1710 cm−1 , that becomes a shoulder in the HA spectra of
Fig. 4. FTIR spectra of HAs isolated from soil and from pile C2 at 3, 61 and 267 days of composting.
the last sampling, and 1460 cm−1 . Simultaneously, the absorption bands at about 1540 and 1507, 1640, 1220 and 1120 cm−1 appear to increase slightly during composting, particularly in the HA fraction from series C2. In general, the results obtained by FT IR spectroscopy confirm those of elemental analysis, and also provide additional information, showing, as composting time progressed, a decrease of aliphatic components and an increase in aromaticity, a greater occurrence of N- and O-containing groups. Altogether, it is interesting to note that, as pointed out in Figs. 3 and 4 where a typical FT IR spectrum of indigenous soil HA is reported for comparison, FT IR spectra of HAs isolated from both series at the final stage of the composting process appear increasingly similar to those of native soil HAs. 3.5. Fluorescence spectra
Fig. 3. FTIR spectra of HAs isolated from soil and from pile C1 at 3, 61 and 267 days of composting.
The fluorescence emission spectra of HAs from C1 and C2 series (Fig. 5, upper) consist with a typical unique broad band with the maximum centered at 440 nm for 1C1- and 1C2-HAs. With increasing composting time, the emission maximum clearly shifts towards longer wavelengths, reaching maximum values at 465 and 467 nm, respectively, in HA-13C1 and HA-13C2. The synchronousscan spectra (Fig. 5, below) of HAs isolated from both series show a similar trend, featuring the presence of numerous peaks at low wavelengths (about 330–390 nm), which disappear with increasing composting time, there was an increased intensity of a peak located in the region of higher wavelengths, 510 and 511 nm, respectively, for HA-13C1 and HA-13C2. According to Senesi et al. [21], the comparative analysis of these spectra indicate, as composting time progresses, an increase in aromatic polycondensation and degree of humification of the raw materials, and at the same time a decrease in initial molecular heterogeneity. As expected, additional information was obtained by total luminescence spectroscopy (TLS), which provides a complete rep-
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Table 3 Major FT IR absorption bands and assignments for HAs examined Wavenumber (cm−1 )
Assignment
3444–3419 3006 2925 and 2854 1743–1745 1710 1640–1651 1540–47 and 1507 1463–1457 1420 1380 1265–1266 1220–1227 1120–1111 1043–1034
O H stretching, N H stretching (minor), hydrogen-bonded OH Stretching of aromatic C-H Asymmetric and symmetric C H stretching of CH2 group C O stretching of aldehydes and ketones C O stretching of COOH Aromatic C C skeletal vibrations, C O stretching of amide groups (amide I band), C O of quinone and/or H-bonded conjugated ketones N-H deformation and C N stretching (amide II band), aromatic C C stretching C H asymmetric bending of CH3 groups O H deformation and C O stretching of phenolic groups COO− antisymmetric stretching, C H bending of CH2 and CH3 groups C O stretching of aryl esters C O stretching of aryl ethers and phenols C O stretching of secondary alcohols C O stretching of polysaccharides or polysaccharide-like substances
resentation of fluorescent spectral features of the sample examined. In particular, TL spectra are the result of merging a series of emission scans from excitations over a range of wavelengths, and provide more detailed information such as the number and type of fluorophores present as well as their abundance. More recently, TLS has been applied successfully to the study of fluorescent compounds
occurring both in dissolved and in stabilized organic matter from various sources and origins [22–26]. Contour maps of HAs isolated for both series at the different stage of composting are shown in Fig. 6. The contour maps of HAs isolated from the initial stage of composting are characterized by the presence of a main fluorophore, identified by the emission–excitation wavelength pair
Fig. 5. Fluorescence emission spectra (upper) and synchronous-scan spectra (lower) of HAs isolated from both series at different stages of composting.
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Fig. 6. TL spectra (contour maps) of HAs isolated from both series at different stages of composting.
(EEWP) 275ex /340–345em , which may be ascribed to simple components of proteinaceous origin such as tryptophan and tyrosine [25,27]. EEMs spectra of HAs isolated from the next stages of composting show the same fluorophores and the appearance of a new peak, identified by the EEWP 310ex /405–420em , which may be associated to aromatic units such as simple phenolic-like, hydroxy-
substituted benzoic and cinnamic acid derivatives [21]. The EEMs spectra of HAs 6C exhibit the peak described above slightly shifted towards longer wavelengths, and an additional one, characterized by the EEWP 370-380ex /460em , likely attributable to the occurrence of scopoletin-like structures, suggesting an increasing degree of oxidation of the composting materials. Finally, EEMs spectra of
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HAs isolated from the last two stages of composting of both series are characterized by the presence of a single peak, centered by the EEWP, 425–430ex /495–500em , which can be associated to the presence of conjugated chinon and phenol units with a high polycondensation degree [21,27]. In general, the observed shift towards higher wavelengths, evident in all fluorescence spectra of HAs examined from both series, could be ascribed to the greater probability of electron transitions between the singlet state and ground state in the molecules occurring in the samples as composting time increases, as a result of their higher molecular complexity, that is, to the presence of a more extended -electron systems [21]. In all cases a slight but constant increase of the fluorescence intensity (FI) values was observed with increasing composting time, especially in the HA fractions of the last sample. The HAs 6C from both series show lower FI values for both fluorophores with respect to those recorded in the other humic fractions, probably because of the relatively greater occurrence of electron-withdrawing groups, such as carboxyl, carbonyl and Schiff-base structures [21], also confirming what was previously indicated by FT IR data. These results indicate that during the composting process the organic substrate is gradually converted into humic-like materials, giving compounds of increasing molecular complexity and structural homogeneity.
of composting showed a progressive stabilization/humification, because of different processes occurring during composting. On the whole, the main transformations concerned a relative increase of proteinaceous materials, an extended degradation of carbohydrates likely due to microbial activity, a decrease of molecular heterogeneity, an increase in unsaturated structures, molecular size, level of conjugated chromophores, aromatic polycondensation, and humification degree. After 9 months, the chemical and spectroscopic characteristics reached by the humic fractions isolated both from pile C1 (moistened with tap water) and pile C2 (moistened with olive oil wastewater) appeared to be more similar to those of soil native HA, making both composts well suited to application onto soil as an amendment. No significant differences were observed between the two treatments at the end of composting, the properties of the HA fractions isolated from the last stage being very similar. It is interesting to note, however, that the different treatments carried out in the first 30 days affected the start of the composting process. In particular, the treatment with tap water seemed to accelerate, (or treatment with olive oil wastewater seemed to slow down), the early mineralization processes.
4. Discussion
This work was financed by the CNRST of Morocco (Project PROTARS D16/22).
The main changes observed in the HAs isolated at different stages during the composting process suggest the following remarks:
References
(a) The N content increase can likely be attributed to both microbial biomass N (proteins, amino sugars) which might be incorporated into the humic substances by biochemical mechanisms and/or physical entrapment [28], and to slight enrichment of N-containing compounds, such as proteinaceous materials, incorporated into HA molecules during their formation [2]. Furthermore, C/H and O/C increases could be related, according to previous studies [2,19], to a progressive reduction of aliphatic material and to an enrichment of O-alkyl material, due to oxidation reactions occurring during the composting process and to the formation of structures stabilized by means of C C linked networks [29]. Additionally, according to Filip and Bielek [30], the C/H increase can be explained with an increase of aromatic structures in the humic acids. (b) The changes observed in the FT IR spectra of the humic acids from the different stages of composting are mainly due both to degradation of the more aliphatic fractions by micro-organisms and to enrichment of COO− and amide structures [10]. These results appear well supported by elemental analysis data. The same variations have been reported by numerous previous works [3,7,10,12,31] for the FT IR spectra of the humic acids obtained by composting organic materials of various origins. (c) The changes observed in fluorescence spectra may be ascribed to a decrease of molecular heterogeneity and an increase of molecular complexity, associated to a greater aromatic character, polycondensation, level of conjugated chromophores, changes produced as composting time increases [3,32,33]. 5. Conclusion The chemical and physico-chemical techniques utilized in this study, such as elemental analysis, and ultraviolet/visible, FT IR and fluorescence spectroscopies, proved to be a powerful tool to monitor the evolution of solid olive mill residues during a composting process. In fact, the humic fractions isolated from the various stages
Acknowledgement
[1] A. D’Annibale, C. Crestini, V. Vinciguerra, G. Giovannozzi Sermanni, The biodegradation of recalcitrant effluents from an olive mill by a whit-rot fungus, J. Biotechnol. 61 (1998) 209–218. [2] A. Roig, M.L. Cayuela, M.A. Sánchez-Monedero, An overview on olive mill wastes and their valorisation methods, Waste Manage. 26 (2006) 960–969. [3] C. Plaza, N. Senesi, G. Brunetti, D. Mondelli, Evolution of the fulvic acid fractions during co-composting of olive oil mill wastewater sludge and tree cutting, Bioresour. Technol. 98 (2007) 1964–1971. [4] M. Papafotiou, M. Phsyhalou, G. Kargas, I. Chatzipavlidis, J. Chronopoulos, Olivemill wastes compost as growing medium component for the production of poinsettia, Sci. Horticult. 102 (2) (2004) 167–175. [5] S. Hachicha, F. Sallemi, K. Medhioub, R. Hachicha, E. Ammar, Quality assessment of composts prepared with olive mill wastewater and agricultural wastes, Waste Manage. (2008) (Epub ahead of print). [6] F. Sellami, R. Jarboui, S. Hachicha, K. Medhioub, E. Ammar, Co-composting of oil exhausted olive-cake, poultry manure and industrial residues of agro-food activity for soil amendment, Bioresour. Technol. 99 (5) (2008) 1177–1188. [7] A. Ouatmane, M.R. Provenzano, M. Hafidi, N. Senesi, Compost maturity assessment using calorimetry, spectroscopy and chemical analysis, Comp. Sci. Util. 8 (2000) 135–146. [8] P. Sequi, A. Benedetti, Management techniques of organic materials in sustainable agriculture, «Integrated plant nutrition system», FAO Fertiliz. Plant Nutr. Bull. (1995) 139–154. [9] U. Tomati, E. Madejon, E. Galli, Evolution of humic acid molecular weight as an index of compost stability, Comp. Sci. Util. 8 (2000) 108–115. [10] G. Ait Baddi, M. Hafidi, V. Gilard, J.C. Revel, Characterization of humic acids produced during composting of olive mill wastes: elemental and spectroscopic analyses (FTIR and 13C-NMR), Agronomie 23 (2003) 661–666. [11] M.R. Provenzano, A. Ouatmane, M. Hafidi, N. Senesi, Differential Scanning Calorimetric analysis of composted materials from different sources, J. Therm. Anal. Cal. 61 (2000) 607–614. [12] M. Fuentes, R. Baigorri, G. González-Gaitano, J.M. García-Mina, The complementary use of 1 H NMR, FTIR and size exclusion chromatography to investigate the principal structural changes associated with composting of organic materials with diverse origin, Org. Geochem. 38 (2007) 2012–2023. [13] F. Sellami, S. Hachicha, M. Chtourou, K. Medhioub, E. Ammar, Maturity assessment of composted olive mill wastes using UV spectra and humification parameters, Bioresour. Technol. 99 (2008) 6900–6907. [14] R.S. Swift, Organic matter characterization, in: D.L. Sparks, A.L. Page, P.A. Helmke, R.H. Loeppert, P.N. Soltanpour, M.A. Tabatabai, C.T. Johnston, M.E. Sumner (Eds.), Methods of Soil Analysis. Part 3. Chemical Methods, SSSA Book Series, No. 5, ASA and SSSA, Madison, WI, 1996, pp. 1011–1069. [15] Y. Chen, N. Senesi, M. Schnitzer, Information provided on humic substances by E4 /E6 ratios, Soil Sci. Soc. Am. J. 41 (1977) 352–358. [16] M. Mustin, Le composte, gestion de la matière organique, Edition Francais Dubus, Paris, 1987. [17] U. Tomati, E. Galli, Bioremediation of olive waste waters by composting, Waste Manage. Res. 13 (1995) 509–518.
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Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Elemental and spectroscopic characterization of humic-acid-like compounds during composting of olive mill by-products Zainab Droussi a , Valeria D’Orazio b , Mohamed Hafidi c , Aaziz Ouatmane a,∗ a
Equipe Environnement et Valorisation des Agro Ressources, BP: 523, FST Beni Mellal, Morocco Dipartimento di Biologia e Chimica Agroforestale e Ambientale, Via Amendola 165/A, 70126 Bari, Italy c Laboratoire d’Ecologie et Environnement, Faculté des Sciences Semlalia, Marrakech, Morocco b
a r t i c l e
i n f o
Article history: Received 22 April 2008 Received in revised form 21 July 2008 Accepted 22 July 2008 Available online 6 August 2008 Keywords: Solid olive mill residues Composting Humic-acid-like Elemental analysis Spectroscopic techniques (Fluorescence FT IR)
a b s t r a c t Humic acids (HAs) were isolated at different stages of composting from two piles of solid olive mill residues (SOR) treated for the first 30 days with tap water (pile C1) or olive mill wastewater (pile C2), for a total composting period of 9 months. The HA fractions were characterized by elemental analysis, UV–visible, Fourier transform infrared and fluorescence spectroscopy in order to monitor humification process and the maturity of the composts. As composting proceeded, the elemental composition of the humic acids showed a decrease in C and H content, and in the C/N ratio, and an increase in N and O contents and in the C/H and O/C ratios. These changes could be attributed to a loss of aliphatic groups and to an increase of aromatic character, polycondensation and degree of oxidation of the HAs. Spectroscopic data agree and support these results, suggesting that the chemical and structural features of the HAs of both composts tend to reach those typical of native soil HAs, that is compounds with a high degree of humification and a high molecular weight and complexity. Therefore, both composting processes seem suitable to produce well-humified organic matter, with important benefits for their use in soil amendment. No differences appeared between the two treatments concerning the humic character of the two final composts. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In most Mediterranean countries, the main processes used for olive oil extraction are still based on the three-phase system which produces enormous quantities of two polluting by-products: olive mill waste water (OMW) and solid olive residues (SOR). These wastes are rich in refractory organic compounds such as phenols, tannins, alcohols, pectin and lipids, which make them very resistant and inappropriate to treatment by conventional biological methods [1]. In general, the most common disposal method for OMW has been limited to evaporation in storage ponds because of the low investment required and the favourable climatic conditions in Mediterranean countries [2]. Nowadays, the composting and/or the co-composting of OMW with SOR represents a successful and suitable method to enhance the quality of organic matter occurring in these materials, with the final production of stable organic compounds that look like native soil organic matter, and particularly humic substances (HS) [3]. Successful application of OMW derived compost both as plant growth medium and plant fertilizer were also
∗ Corresponding author. Tel.: +212 23 48 51 12; fax: +212 23 48 52 01. E-mail address: [email protected] (A. Ouatmane). 0304-3894/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2008.07.136
reported in several recent works [4–6]. Obviously, the evolution of organic matter stabilization during composting is of primordial importance in controlling the efficiency of the process. With this purpose, several physical, chemical and biological techniques and parameters have been proposed [7], and recently compost maturity has been investigated by monitoring the humification degree of these materials through chemical and physico-chemical analyses. Sequi and Benedetti [8] proposed the use of the humification parameters (index, rate and degree of humification) and Tomati et al. [9] suggested monitoring the molecular weight of the humic substances as an index of stability. Advanced physico-chemical techniques such as Fourier transform infrared (FT IR), Fluorescence, and Nuclear Magnetic Resonance (13 C NMR) spectroscopies and thermal analysis, such as Differential Scanning Calorimetry (DSC), have also been used to characterize organic matter of composts and humic substances originating from olive mill wastes [10–12], showing that the composting technique represents a suitable treatment for promoting organic matter humification in these materials, thus improving their agronomic value and minimizing possible negative environmental impact. In recent work good correlations were also observed between common humification indices including the E4/E6 absorption ratio of humic substances and the agronomic value of composts from olive by-products [13]. However, although a wide literature is available on the composting of OMW, relatively
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little information is available on the composting of SOR, and, particularly, on the evolution of humic substances during the composting of these materials. The aim of the present study was to follow the evolution occurring in the humic acid-like fraction during the composting of SOR and their co-composting with OMW by means of chemical and spectroscopic analyses.
Fisher’s least significant difference (LSD) protected t-test. The confidence intervals used were 95, 99, and 99.9%. The symbols used to indicate the P values are † significant at P ≤ 0.05, †† significant at P ≤ 0.01, and ††† significant at P ≤ 0.001. Furthermore, Student’s t-test was applied to evaluate the differences between the HA fractions isolated at the same composting time in the different piles. In this case the symbols used to indicate the P values are *significant at P ≤ 0.05, **significant at P ≤ 0.01, and ***significant at P ≤ 0.001.
2. Materials and methods 2.5. E4 /E6 ratio 2.1. Composting process For both the composting and the co-composting trial, a pile was prepared using 1.5 tonnes of olive oil extraction residues obtained from a three-phase system olive mill in Morocco. During the first 30 days the composting pile (Pile C1) was moistened with tap water while the co-composting pile (Pile C2) was moistened with a total of 0.25 tonnes of olive oil wastewater. The composting process was performed for 267 days during which the piles were forked over periodically and their humidity was maintained at around 50–60%. Biological activity was monitored by measuring the temperature of the piles at a depth of 30 cm. To follow the evolution of the humicacid-like fraction, composite samples were collected at different points from the top to the bottom of the plies after: 3 days (samples 1C1 and 1C2), 31 days (samples 3C1 and 3C2), 61 days (samples 6C1 and 6C2), 195 days (samples 10C1 and 10C2) and 267 days (samples 13C1 and 13 C2). The composite samples were homogenized and immediately stored at −18 ◦ C until analysis. 2.2. Humic acids The HAs were isolated on the basis of their solubility in water as a function of pH, with some modification with respect to the procedure of the International Humic Substances Society (IHSS) [14]. Briefly, to 30 g of each compost sample, previously air-dried and crushed, was added 100 mL 0.1 M KOH. The mixtures were shaken mechanically for 2 h at room temperature (RT, 293 + 2 K), then centrifuged at 5000 rpm for 25 min. This operation was repeated until the supernatant obtained was clear. The combined alkaline supernatants were acidified with 6N H2 SO4 (pH 2), allowed to stand for 24 h at 4 ◦ C, to permit coagulation of the HA fractions, then centrifuged again at 5000 rpm for 25 min. The HA precipitates were purified by dissolution in a minimal volume of 0.1 M KOH, transferred to a dialysis membrane (SpectraPore membrane, MWCO 1000 Daltons) and dialyzed against double distilled water in order to eliminate excess salts. Finally, HAs were freeze-dried for 72 h. Moisture and ash contents were measured by heating the HAs overnight at 105 ◦ C and for 2 h at 550 ◦ C, respectively. 2.3. Elemental analysis (C, H, N, S) Carbon, hydrogen, nitrogen, and sulphur concentrations were determined using a combustion-gas chromatography technique (Fisons EA 1108 Elemental Analyzer, Milan, Italy). The oxygen content was calculated by difference: O% = 100 − (C + H + N + S)%. The instrument was calibrated using a BBOT [2,5-bis-(5-tert-butylbenzoxazol-2-yl)-thiophen] standard (ThermoQuest Italia s.p.a.). The data obtained were corrected for moisture and ash contents. All HA samples were analyzed in triplicate. 2.4. Statistical analysis The elemental composition of HA fractions isolated during the composting time from each pile were statistically analyzed using one-way analysis of variance (ANOVA, F-distribution), followed by
The E4 /E6 ratio was calculated as the ratio of absorbance at 465 and 665 nm measured by a PerkinElmer model Lambda 15 UV–vis spectrophotometer on solutions of 3.0 mg of each HA dissolved in 10 mL of 0.05 M NaHCO3 , with the pH adjusted to 8.3 with NaOH. The E4 /E6 ratio is very useful a spectroscopic index to evaluate the different sources of humic material [15]. In particular, the E4 /E6 ratio is inversely related to molecular weight, C content, aromaticity and polycondensation degree of the organic molecules, whereas a positive correlation has been found between this ratio and O and carboxylic group contents and aliphaticity [15]. 2.6. Fourier transform InfraRed (FT IR) spectroscopy The FT IR spectra were recorded on pellets obtained by pressing under reduced pressure a mixture of 1 mg of HA and 400 mg of dried spectrometry grade KBr using a Nicolet Nexus FT IR spectrophotometer equipped with Nicolet Omnic 6.0 software. Spectra were recorded in the range 4000–400 cm−1 with 2 cm−1 resolution, and 64 scans were performed on each acquisition. 2.7. Fluorescence spectroscopy Fluorescence spectra were recorded on aqueous solutions of 100 mg L−1 HA after overnight equilibration at room temperature, and adjustment to pH 8.0 with 0.05 mol L−1 NaOH, using a Hitachi model F-4500 luminescence spectrophotometer. One-dimensional emission spectra were recorded over the range 380–550 nm at a constant excitation wavelength of 360 nm. Excitation spectra were recorded over the range 300–500 nm at a fixed emission wavelength of 520 nm. Synchronous-scan excitation spectra were measured by simultaneously scanning both the excitation and the emission wavelengths (from 300 to 550 nm), while maintaining a constant, optimized wavelength difference = em − ex = 18 nm. The Total Luminescence (TL) spectra were obtained in the form of excitation/emission matrix (EEM, or contour maps) by scanning the wavelength emission over the range 300–600 nm, while the excitation wavelength was increased sequentially in 5 nm steps from 250 to 500 nm. The EEM spectra were generated from TL spectral data by using Noesys 2.4 software. The fluorescence intensity (FI) values (in arbitrary units) were normalized using a quinine sulphate standard (10 mol in 0.1N H2 SO4 ). 3. Results 3.1. Temperature evolution Temperature evolution during the composting process corresponds to a typical curve of an optimal aerobic composting system [16,17]. In both piles, the temperature increased in a few days from 25 to 60 ◦ C (Fig. 1), and oscillated thereafter between 55 and 65 ◦ C for approximately 2 months. This confirms the high biodegradability of SOR and their ability to produce sufficient heat during composting to ensure the hygienisation of the compost. A similar temperature evolution was reported during composting of OMW
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Fig. 1. Temperature evolution during composting of pile C1 (SOR moistened with tap water) and pile C2 (SOR moistened with OMW).
by Ait Badi et al. [10] and Hachicha et al. [5]. The end of the thermophilic phase was reached after approximately 9 months for the two piles. At this stage both composts could be considered stable. Comparison between the two piles showed that primary addition of OMW to SOR did not affect temperature evolution during the composting experiment. Similar results were reported by Hachicha et al. [5] for a mixture of exhausted olive cake and poultry manure compost that was moistened with OMW during the first 2 months of composting. In contrast, the negative effects on microbial activity of adding OMW, and hence the effects on temperature increases during organic waste composting, have been observed by others authors [18]. The results obtained in the present work could be attributed to the low amounts of OMW used. 3.2. Elemental analysis The elemental compositions of the HAs isolated from the two composts at different stages of the composting process are shown in Table 1. Data reported indicate, for both series, a decrease in C content as composting progresses, but some differences between the two piles are evident with regard to the sampling interval examined. In particular, careful analysis of the composition of HA from series C1 indicates that, with respect to the HA 1C1, the HA 3C1 samples show a significantly lower (P < 0.001) C content, whereas no difference was found in the next sample HA 6C1, as if to suggest a slowdown of the process. The decrease was significantly lower (P < 0.001) in sample HA 10C1 and finally it remained unchanged in HA 13C1. The C content of the HA fractions isolated from the C2 series, initially similar to that of the other series, did not show differences between the HA isolated from the first two samples, then decreased during the whole composting process. In particular, the reduction was about the 4% in samples HA 6C2 (P < 0.01), enhanced markedly (by about 10%) in HA 10C2 (P < 0.001), and fell
slightly (about 3%) in HA 13C2 (P < 0.05) (Table 1). The H content shows a similar trend (Table 1). The N content exhibited the opposite tendency, that is it increased during the whole composting process in the HA fractions of both series, showing a value significantly higher (P < 0.001) in each HA fraction with respect to that of the corresponding previous one. The only exception was sample HA 13C1, characterized by a significant increase at the lower confidence interval (P < 0.01). Similarly, the O content results were significantly higher (P < 0.01) in the HA 3C1 with respect to HA 1C1, unchanged in the next sample HA 6C1, and again increased in the sample HA 10C1. Finally, a slight decrease (P < 0.05) was observed in the HA 13C1 fraction (Table 1). In contrast, the O content of the HA fractions from the C2 series remained unchanged until the third sampling, when it increased significantly (P < 0.001) in HA 10C2, and decreased slightly (P < 0.05) in HA 13C2. Furthermore, with the exception of the first sample, the O content results were always greater, at various confidence intervals, in the HAs from the C1 series with respect to the corresponding HAs from the C2 series. Finally, the S content increased significantly (P < 0.001) in the HA fractions of the C1 series, whereas in the C2 series it was significantly enhanced (P < 0.001) only in the HA fractions isolated from samples 10C2 and 13C2. To better evaluate the evolution of the HA fractions during the composting process as a function of the two different treatments, Student’s t-test was applied to analyze the elemental composition of the corresponding HA samples. For all elements, the HA fractions from to 31 days of composting time result those with the greater differences between the two piles (P < 0.001) (Table 1). In particular, HA 3C2 shows higher C and H contents and lower N, S and O contents with respect to the corresponding HA 3C1. Furthermore, the N content of HA 1- and HA 3C2 was significantly lower and HA 6-, HA 10- and HA 13 C2 significantly higher, with respect to the corresponding HA fractions from pile C1 (Table 1). Additional slight differences are also evident at various
Table 1 Elemental composition (moisture and ash free) of humic acids isolated from pile C1 and pile C2 at different stages of composting Composting time (days)
3 31 61 195 267
C%
N%
H%
S%
O%
C1
C2
C1
C2
C1
C2
C1
C2
C1
C2
70.00 64.63††† 63.38 57.14††† 57.81
70.58 70.02*** 67.12††* 60.47†††* 58.47†*
1.05 2.44††† 2.74††† 5.17††† 5.35††
0.99*** 1.30†††*** 3.40†††*** 5.76†††** 5.43†††*
10.29 8.70††† 8.41† 6.32††† 6.15
10.45 10.34*** 9.10†††* 6.23††† 6.34
0.97 2.46††† 1.48††† 2.60††† 4.47†††
0.68** 1.14*** 1.37* 2.78††† 7.04†††*
17.69 21.77††** 23.99* 28.77††* 26.22†***
17.30 17.20 19.01 24.76††† 22.72†
† †† , and ††† (LSD test) refer to significant differences: P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001 respectively; *, **, and *** (t-test) refer to significant differences: P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001 respectively.
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Table 2 Atomic ratios and E4 /E6 ratios of humic acids isolated from pile C1 and pile C2 at different stages of composting Composting time (days)
3 31 61 195 267
C/N
C/H
O/C
E4 /E6
C1
C2
C1
C2
C1
C2
C1
C2
77.48* 30.88*** 27.02*** 12.89*** 12.60
83.26 62.69 23.05 12.26 12.57
0.57 0.62*** 0.63* 0.75** 0.78
0.56 0.56 0.61 0.81 0.77
0.19 0.25*** 0.28* 0.38* 0.34***
0.18 0.18 0.21 0.31 0.29
2.0 2.9 3.4 6.2 5.7
2.2 2.6 3.4 5.8 6.1
*, **, and *** (t-test) refer to differences significant to P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001 respectively.
confidence intervals between the other HA fractions isolated from the two piles during the whole composting period. In general for both series, with increasing composting time, C/N ratios decreased, whereas C/H and O/C ratios increased (Table 2). To evaluate the differences between the atomic ratios of HA fractions in the two piles Student’s t-test was applied and the corresponding P values are reported in Table 2. The HAs from series C1 showed a lower C/N ratio, at various confidence intervals, with respect to the HAs from C2, reaching similar values only in the last sample (Table 2). The C/H ratio was characterized by similar values in the HA fractions of the first and last samples from both series, whereas it was higher in the HA-3C1 and -6C, and lower in HA 10C1 than in the C2 series (Table 2). Finally, the O/C ratios, with the exception of HA samples from the first sample, were always higher in the HAs from series C1 with respect to the corresponding HAs series C2 (P values in Table 2). In general, the extent of these variations followed the trend described for the corresponding elemental composition, at the end
of the composting process reaching similar values for C/N and C/H ratios, whereas a greater O/C ratio was observed in sample HA 13C1 with respect to HA 13C2 (Table 1). As previously reported [2,9,19,20] these values indicate that, with increasing composting time, N-containing compounds probably increased because proteinaceous materials are incorporated into the HA, and, simultaneously both the oxidation and the proportion of unsaturated structures with respect to saturated structures increased (lower C/H ratios). 3.3. E4 /E6 ratio The E4 /E6 ratios of HAs from both series were similar at the beginning of the composting process, then showed a constant increase with composting time, reaching comparable values in the HAs isolated from the last two stages (10C1 and 13C1, 10C2 and 13C2 samples). In particular, for the HA samples from series C1 the E4 /E6 ratio was markedly enhanced in the first 30 days of composting, reaching a maximum value in the sample HA 10C1. A slight decrease was then observed in HA 13 C1 (Table 2). In contrast, the E4 /E6 ratio of HAs from series C2 showed a more gradual increase as composting progressed, reaching a maximum value in sample HA 13C2 (Table 2). A previous study [15] suggested that the E4 /E6 ratio of HAs is correlated positively with the O content and degree of oxidation, and negatively with C content, aromaticity, polycondensation and molecular weight. In agreement with this study, the increase measured correlated positively to O content and negatively to C content. To verify these relationships between spectroscopic properties and elemental composition of HAs, a linear regression was performed by correlating E4 /E6 data with O and C contents (Fig. 2). As expected, in both cases we found the coefficient of determination values (r2 ) very close to 1, espe-
Fig. 2. Linear regression with corresponding r2 values between, respectively, E4 /E6 ratios and O% (upper) and E4 /E6 ratios and C% (lower) of HAs isolated from both series at different stages of composting.
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cially in the linear regression E4 /E6 vs. C content (r2 values slightly higher). 3.4. FT IR spectra The FT IR spectra of a typical native soil HA and HA fractions 1C1, 6C1 and 13C1 and 1C2, 6C2 and 13C2, are shown in Figs. 3 and 4 respectively. The main absorption bands and corresponding assignments are summarized in Table 3. All spectra feature common and distinctive absorption bands, with some differences in their relative intensity. The main characteristics of these spectra are the following: about 3400 cm−1 (OH stretching and, secondarily, N H stretching of various functional groups); about 3006 cm−1 (stretching of aromatic C H); about 2925 and 2853 cm−1 (asymmetric and symmetric C H stretching respectively of CH2 groups); about 1743 cm−1 (C O stretching of aldehydes and ketones); about 1714 cm−1 (C O stretching of COOH and other carbonyl groups); about 1640 cm−1 (aromatic C C skeletal vibrations, C O stretching of quinone and amide groups (amide I band), C O of H-bonded conjugated ketones); about 1540 and 1507 cm−1 (preferentially ascribed to N H deformation and C N stretching of amides (amide II band); about 1460 cm−1 (C H bending of CH3 groups); about 1420 cm−1 (O H deformation and C O stretching of phenolic OH); about 1380 cm−1 (C H deformation of CH2 and CH3 groups, and/or antisymmetric stretching of COO− groups); about 1265 cm−1 (C O stretching of aryl esteres); about 1220 cm−1 (C O stretching of aryl ethers and phenols); about 1120 cm−1 (C O stretching of secondary alcohols and/or ethers); and, finally, about 1040 cm−1 (C O stretching of polysaccharides or polysaccharide-like substances). With increasing composting time, FT IR spectra of HAs from both series show the disappearance of bands at 3006 and 1743 cm−1 , and a decrease in the relative intensity of absorption bands at 2925 and 2853 cm−1 , 1710 cm−1 , that becomes a shoulder in the HA spectra of
Fig. 4. FTIR spectra of HAs isolated from soil and from pile C2 at 3, 61 and 267 days of composting.
the last sampling, and 1460 cm−1 . Simultaneously, the absorption bands at about 1540 and 1507, 1640, 1220 and 1120 cm−1 appear to increase slightly during composting, particularly in the HA fraction from series C2. In general, the results obtained by FT IR spectroscopy confirm those of elemental analysis, and also provide additional information, showing, as composting time progressed, a decrease of aliphatic components and an increase in aromaticity, a greater occurrence of N- and O-containing groups. Altogether, it is interesting to note that, as pointed out in Figs. 3 and 4 where a typical FT IR spectrum of indigenous soil HA is reported for comparison, FT IR spectra of HAs isolated from both series at the final stage of the composting process appear increasingly similar to those of native soil HAs. 3.5. Fluorescence spectra
Fig. 3. FTIR spectra of HAs isolated from soil and from pile C1 at 3, 61 and 267 days of composting.
The fluorescence emission spectra of HAs from C1 and C2 series (Fig. 5, upper) consist with a typical unique broad band with the maximum centered at 440 nm for 1C1- and 1C2-HAs. With increasing composting time, the emission maximum clearly shifts towards longer wavelengths, reaching maximum values at 465 and 467 nm, respectively, in HA-13C1 and HA-13C2. The synchronousscan spectra (Fig. 5, below) of HAs isolated from both series show a similar trend, featuring the presence of numerous peaks at low wavelengths (about 330–390 nm), which disappear with increasing composting time, there was an increased intensity of a peak located in the region of higher wavelengths, 510 and 511 nm, respectively, for HA-13C1 and HA-13C2. According to Senesi et al. [21], the comparative analysis of these spectra indicate, as composting time progresses, an increase in aromatic polycondensation and degree of humification of the raw materials, and at the same time a decrease in initial molecular heterogeneity. As expected, additional information was obtained by total luminescence spectroscopy (TLS), which provides a complete rep-
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Table 3 Major FT IR absorption bands and assignments for HAs examined Wavenumber (cm−1 )
Assignment
3444–3419 3006 2925 and 2854 1743–1745 1710 1640–1651 1540–47 and 1507 1463–1457 1420 1380 1265–1266 1220–1227 1120–1111 1043–1034
O H stretching, N H stretching (minor), hydrogen-bonded OH Stretching of aromatic C-H Asymmetric and symmetric C H stretching of CH2 group C O stretching of aldehydes and ketones C O stretching of COOH Aromatic C C skeletal vibrations, C O stretching of amide groups (amide I band), C O of quinone and/or H-bonded conjugated ketones N-H deformation and C N stretching (amide II band), aromatic C C stretching C H asymmetric bending of CH3 groups O H deformation and C O stretching of phenolic groups COO− antisymmetric stretching, C H bending of CH2 and CH3 groups C O stretching of aryl esters C O stretching of aryl ethers and phenols C O stretching of secondary alcohols C O stretching of polysaccharides or polysaccharide-like substances
resentation of fluorescent spectral features of the sample examined. In particular, TL spectra are the result of merging a series of emission scans from excitations over a range of wavelengths, and provide more detailed information such as the number and type of fluorophores present as well as their abundance. More recently, TLS has been applied successfully to the study of fluorescent compounds
occurring both in dissolved and in stabilized organic matter from various sources and origins [22–26]. Contour maps of HAs isolated for both series at the different stage of composting are shown in Fig. 6. The contour maps of HAs isolated from the initial stage of composting are characterized by the presence of a main fluorophore, identified by the emission–excitation wavelength pair
Fig. 5. Fluorescence emission spectra (upper) and synchronous-scan spectra (lower) of HAs isolated from both series at different stages of composting.
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Fig. 6. TL spectra (contour maps) of HAs isolated from both series at different stages of composting.
(EEWP) 275ex /340–345em , which may be ascribed to simple components of proteinaceous origin such as tryptophan and tyrosine [25,27]. EEMs spectra of HAs isolated from the next stages of composting show the same fluorophores and the appearance of a new peak, identified by the EEWP 310ex /405–420em , which may be associated to aromatic units such as simple phenolic-like, hydroxy-
substituted benzoic and cinnamic acid derivatives [21]. The EEMs spectra of HAs 6C exhibit the peak described above slightly shifted towards longer wavelengths, and an additional one, characterized by the EEWP 370-380ex /460em , likely attributable to the occurrence of scopoletin-like structures, suggesting an increasing degree of oxidation of the composting materials. Finally, EEMs spectra of
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HAs isolated from the last two stages of composting of both series are characterized by the presence of a single peak, centered by the EEWP, 425–430ex /495–500em , which can be associated to the presence of conjugated chinon and phenol units with a high polycondensation degree [21,27]. In general, the observed shift towards higher wavelengths, evident in all fluorescence spectra of HAs examined from both series, could be ascribed to the greater probability of electron transitions between the singlet state and ground state in the molecules occurring in the samples as composting time increases, as a result of their higher molecular complexity, that is, to the presence of a more extended -electron systems [21]. In all cases a slight but constant increase of the fluorescence intensity (FI) values was observed with increasing composting time, especially in the HA fractions of the last sample. The HAs 6C from both series show lower FI values for both fluorophores with respect to those recorded in the other humic fractions, probably because of the relatively greater occurrence of electron-withdrawing groups, such as carboxyl, carbonyl and Schiff-base structures [21], also confirming what was previously indicated by FT IR data. These results indicate that during the composting process the organic substrate is gradually converted into humic-like materials, giving compounds of increasing molecular complexity and structural homogeneity.
of composting showed a progressive stabilization/humification, because of different processes occurring during composting. On the whole, the main transformations concerned a relative increase of proteinaceous materials, an extended degradation of carbohydrates likely due to microbial activity, a decrease of molecular heterogeneity, an increase in unsaturated structures, molecular size, level of conjugated chromophores, aromatic polycondensation, and humification degree. After 9 months, the chemical and spectroscopic characteristics reached by the humic fractions isolated both from pile C1 (moistened with tap water) and pile C2 (moistened with olive oil wastewater) appeared to be more similar to those of soil native HA, making both composts well suited to application onto soil as an amendment. No significant differences were observed between the two treatments at the end of composting, the properties of the HA fractions isolated from the last stage being very similar. It is interesting to note, however, that the different treatments carried out in the first 30 days affected the start of the composting process. In particular, the treatment with tap water seemed to accelerate, (or treatment with olive oil wastewater seemed to slow down), the early mineralization processes.
4. Discussion
This work was financed by the CNRST of Morocco (Project PROTARS D16/22).
The main changes observed in the HAs isolated at different stages during the composting process suggest the following remarks:
References
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