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Structural study of the fulvic fraction during composting of activated sludge–plant matter: Elemental analysis, FTIR and 13C NMR
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Bioresource Technology 99 (2008) 1066–1072
Structural study of the fulvic fraction during composting of activated sludge–plant matter: Elemental analysis, FTIR and 13C NMR A. Jouraiphy a, S. Amir c, P. Winterton e, M. El Gharous a, J-C. Revel d, M. Hafidi
b,*
a Labo. Fertilite´ des Sols, Centre Arido-Culture, INRA Settat, Morocco Equipe Ecologie Ve´ge´tale, Sol et Environnement, Faculte´ des Sciences Semlalia, De´partement de Biologie, BP/2390 Marrakech, Morocco c De´partement de Biologie, Faculte´ Polydisciplinaire, Beni Mellal, Morocco Equipe Agronomie, Environnement et Ecotoxicologie (A2E), Ecole Nationale Supe´rieure Agronomique, Auzeville-Tolosane, BP/107 Toulouse, France e Universite´ Paul Sabatier, Toulouse, France b
d
Received 25 April 2006; received in revised form 26 February 2007; accepted 27 February 2007 Available online 18 April 2007
Abstract The starting fulvic structures isolated from an initial mixture of activated sludge and plant matter presented abundant peptide structures and hydrocarbons that absorb in FTIR spectra around (1650 and 1560 cm 1) and 1072 cm 1, respectively. They also present a high resonance signal in the O- and N-alkyl areas of 13C NMR spectra. As composting proceeded, some changes led to the formation of the molecular structures of fulvic fraction as demonstrated by a decrease of intensity of compounds absorbing around 1072 cm 1 and an increase of those absorbing around 1140 cm 1. The resonance of O- and N-substituted alkyl carbon also decreased from 55.7% to 33.8%, with an increase in the intensity of aromatic carbons, alkyls and carboxyls. These data indicate that the microbial community that developed during composting used polysaccharides as an energy source, structures which are supplied in abundance in the initial material. The fulvic fraction of the final compost is much richer in aromatic structures and aliphatic ethers/esters, which are most likely preserved from the original material but probably also synthesized through the microbial activities. The occurrence of alkyl ethers/esters at the end of composting is demonstrated by strong absorbance around 1140 cm 1 in the FTIR spectra and large peaks at 32 and 174 ppm in the NMR spectra. These structures could also be produced following the creation of ether/ester bonds during the humification process. 2007 Elsevier Ltd. All rights reserved. Keywords: Fulvic fraction; Compost; Polysaccharides; Resistant aliphatic; Aromaticity
1. Introduction For several decades, the increases in agricultural production have been closely linked to the use of chemical fertilizers (Wagner, 1999; Ouatmane, 2000). These fertilizers, however beneficial to productivity, contribute to the pollution of food chains and of water tables. To compensate for these risks and with a view to enriching soil impoverished by erosion and the intensification of farming, interest in
*
Corresponding author. E-mail address: hafi[email protected] (M. Hafidi).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.02.031
recycling organic waste has gained momentum (Kavira and Sharma, 2003). Several techniques are proposed in the scientific literature to deal with this waste (e.g. incineration, controlled tipping). However, most of these techniques are costly, require specialist personnel and have limitations (Amir, 2005; Lguirati et al., 2005). Therefore, composting is a more accessible technique for recycling organic waste and transforming it into an ecologically stable product agronomically beneficial for the soil (Ouatmane et al., 2000; Jouraiphy et al., 2005). However, the success of the composting process is gauged by the quality of the final product, especially its stability. In this context, numerous studies have been carried out to guarantee the
A. Jouraiphy et al. / Bioresource Technology 99 (2008) 1066–1072
stability and maturity of composts, using several different ways to approach the problem: observation of empirical evidence, physicochemical or microbiological studies, spectroscopy or humic examination (Mathur et al., 1993; Amir, 2005). Some works (Inbar et al., 1992) have revealed that the maturity of compost is closely linked to the levels of humic substances formed during the final stages of the composting process. However, the heterogeneity of these substances presents a serious obstacle to the determination of their exact chemical structure as well as to learning about the processes involved in their formation (Hayes et al., 1989). In general, humic substances are composed of a very large variety of aromatic rings (phenolic and quinonic) bound by acid functional groups or by peripheral aliphatic chains (polysaccharides, peptides, etc.) and grouped into the different molecular arrangements that make up the structure of humic substances (Piccolo, 2002; Jarde et al., 2003; Amir et al., 2006). These can be divided mainly into humic and fulvic fractions). Despite the quantity of works on the subject of humic substances, information about the fulvic fraction remains limited, probably because of their chemical complexity (Da Silva et al., 1998; Joaquim et al., 1998; Ait Baddi et al., 2004; Hafidi et al., 2005a). The objective of this study is to gain a better understanding of the humification process and the formation of fulvic structures throughout the different stages of composting of activated sludge. Various complementary techniques, such as elemental analysis, Fourier transform infra-red spectroscopy (FTIR) and carbon 13 Nuclear Magnetic Resonance (13C NMR) were all used at the different stages of the composting process. 2. Methods
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to be distinguished. The stabilization phase where the average temperature of the heap raises to 72 C within a matter of days. The maturation phase presenting a steady fall in temperature as it slowly reaches equilibrium with ambient temperature (Jouraiphy et al., 2005). Samples of compost were taken at different stages of the process from raw mixture, after 30, 60 and 135 days. The sampling was done at each stage from different places within the heap (center, surface, core of the heap, sides) and after thorough mixing of the various sub-samples, a representative 500 g portion was collected and refrigerated until analysis. Table 1 shows the variations of certain physicochemical properties of the mixture at different stages of the composting process. 2.2. Extraction of fulvic fraction The fulvic fraction was extracted from a fresh 30 g sample. It was defatted using 120 ml of a chloroform–methanol solvent mixture (2:1) (Lichtfouse et al., 1998). This step was repeated three times at 4 C. The solvent was then evaporated off and the samples were treated three times with 40 ml of distilled water so as to extract the water-soluble non-humic substances (sugars, proteins). Then, the humic substances were extracted with 40 ml 0.1 M NaOH. This was repeated several times until the extract obtained was colourless. After filtration, the solutions were pooled and the humic acids were then precipitated out of solution with 1.5 M H2SO4 for 24 h at 4 C. The solution containing the fulvic fraction was dialyzed with a Spectra-Por membrane (1000 Da) to eliminate excess salts. The content of fulvic structures was then calculated after freeze drying. 2.3. Elemental analysis
2.1. Composting The activated sludge was taken from the aerobic waste water treatment plant of Khouribga (Morocco). Dried sludge (10 m3) was mixed with 5 m3 of fresh plant matter and left in a prism-shaped heap (L = 8 m · h = 1.5 m) on a composting platform. The mixture was prepared so as to have the optimal parameters for good composting, namely 60% humidity and a C/N ratio of around 30. The heap was turned every fortnight to maintain good aerobic conditions and was watered. The temperature of the compost heap was checked every day in several locations. The temperature data enables two successive phases
Elemental analysis was carried out using a Carbograph Autoanalyser (Fison Carlo Erba EA 1110) to analyse C, H and N and a type EA 1106 instrument to analyse the O of the humic acids. 2.4. Fourier transform infra-red spectroscopy Two mg of fulvic fraction extracted from each compost sample were mixed with 250 mg of KBr. The absorption spectra of the pellets were recorded on a Perkin Elmer 1600 FTIR spectrophotometer from 4000 to 400 cm 1 at 16 nm/s.
Table 1 Variation of the physico-chemical characteristics of composted sewage sludge and plant matter Stage
pH
C/N
Dec%a
Humic fraction (g kg
Raw mixture 30 days 60 days 135 days
6.36 ± 0.24 5.7 ± 0.18 6.1 ± 0.33 6.23 ± 0.12
29.75 25.98 23.76 12.15
– 6.6 ± 0.33 29.3 ± 0.58 60.8 ± 0.68
26.5 ± 1.55 30.0 ± 1.39 39.7 ± 0.47 39.5 ± 0.27
a
% Dry weight; Dec%: % decomposition; HA: humic acids.
1
dry matter)
Fulvic fraction (g kg 38.2 ± 0.97 22.2 ± 1.46 11.4 ± 2.4 17.8 ± 1.3
1
dry matter)
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2.5. Carbon-13 nuclear magnetic resonance The samples were prepared by dissolving 100 mg of fulvic fraction in 3 ml of NaOD/D2O (0.5 M). The 13C NMR spectrum was recorded with a Bruker ES-WB 300 spectrometer working at 75.469 MHz. A gated pulse decoupling technique was used to suppress any increase in signal intensity due to the Nuclear Overhauser effect. The spectrum was recorded in the following conditions: relaxation: 1.8 s, acquisition period: 0.98 s, the pulse: 35. Total acquisition required 72 h. The areas of the spectra that were integrated were 0–55 ppm (aliphatic carbon), 55– 110 ppm (aliphatic carbon substituted with O or N), 110– 165 ppm (aromatic carbon), and 165–200 ppm (carboxylic carbon). 3. Results and discussion During the stabilisation phase of composting of activated sludge and plant matter, the presence of easily broken down substances allows intense microbial activity, which raises the average temperature of the heap to 72 C in the first days of composting. Secondly comes the maturation phase where temperature decreases because the remaining organic matter (mainly lignocellulose compounds) resists microbiological attack. The C/N ratio and the degree of decomposition reached 12% and 60% respectively after 135 days of composting the activated sludge–plant matter mixture (Table 1). The results illustrate successful composting: similar results have been found in other studies (Zorpas et al., 2003; Zbytniewski and Buszewski, 2005). Compost maturity has also been indicated by the increase in the level of humic acids within the compost (Table 1). Numerous authors have defined the maturity of composts by their level of humic substances: ‘‘humic and fulvic fractions’’. However, in this work, the level of fulvic fraction decreased in the course of composting (Table 1). Some authors suggest that fulvic fraction degradation products are precursors for the formation of humic acids (Doane et al., 2003). Generally, the microbial communities that develop during the composting acquire their energy from degradation of the substances most easily broken down and this could include the compounds that make up the fulvic structures. The biooxidation of these compounds leads to the production of substances with high levels of stable structures, which indicate compost maturity.
Hence the importance of studying in greater depth the evolution of fulvic structures in the course of composting. The correlations between the data gained from the three techniques used: elemental analysis; FTIR; 13C NMR have been commonly investigated to determine the structures composing the humic substances and their evolution through the advancement of the humification process in the course of composting (McDonnell et al., 2001). See Table 2 for the elemental composition and atomic ratios of the fulvic fraction extracted from the compost of activated sludge and plant matter at the various stages of composting. The structures of the fulvic fraction isolated from the uncomposted starting material, compared to the elemental composition of soil (Schnitzer, 1978), show their high content in O, N and O/C ratio; but a low C/H and C/N ratios of about 0.5 and 9.5, respectively. This indicates their richness in aliphatic structures with high O and N contents. Senesi et al. (1996), based on the review of numerous studies of FA in sludge, suggest that lower C/N and C/H ratios may be attributed to the high content of protein decomposition products and aliphatic components respectively in sludge fulvic fraction. In fact, the FTIR spectra of raw mixtures of activated sludge and plant matter exhibit high absorbance at 1650 and 1560 cm 1, attributed to aromatics and peptides, and around 1124 and 1072 cm 1 assigned to the aliphatic structures with high levels of functional groups rich in oxygen (Fig. 1). Similar results were found by Malcolm (1990) in the IR spectra of fulvic acids extracted from sludge, characterized by the presence of aliphatic and aromatic compounds as well as by the breakdown products of the proteins and polysaccharides. The 13C NMR spectra of raw mixture also exhibited strong resonance of oxygen- or nitrogen-substituted alkyl carbons that present amides at 62 ppm, and a strong signal around 71.29 ppm assigned to hydrocabons and anomeric carbon at 101.11 ppm, and many functional entities such as hydroxyls (168.86 ppm) and carboxyls (174.87 ppm) (Fig. 2). Calculations of the areas under the curves of the regions of aliphatic, aromatic and carboxylic carbons are shown in Table 3. The resonance signal in O- and N-alkyl areas presents about of 55.7%. Piccolo et al. (1992) attributed these nitrogen and oxygen-rich compounds to the high levels of non-humified biomolecules (polysaccharides and polypeptides).
Table 2 Elemental composition and atomic ratios of the fulvic fraction at different stages of the composting process of the sludge/fresh plant waste mixture Stages of the composting process
Raw mixture 30 days 60 days 135 days Fulvic acids from soila a
C (%)
35.10 34.61 33.47 31.21 45.70
Fulvic acid of soil model (Schnitzer, 1978).
N (%)
4.33 4.28 4.15 4.21 2.10
H (%)
5.50 5.21 5.26 4.55 5.40
O (%)
44.81 39.00 38.00 44.30 44.80
Ash (%)
10.26 16.90 19.12 15.73 –
Atomic ratios C/N
C/H
O/C
9.46 9.43 9.41 8.65 25.50
0.53 0.55 0.53 0.57 0.70
0.96 0.85 0.85 1.06 0.70
A. Jouraiphy et al. / Bioresource Technology 99 (2008) 1066–1072
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Fig. 1. FTIR spectra of the fulvic fraction extracted from composted activated sludge during the composting process.
At the end of the composting period, the carbon and hydrogen content of the fulvic fraction diminished by about 11% and 17% of the initial amount, respectively
(Table 2). No notable changes were observed in the N content, but the atomic ratio C/N decreased. While, the oxygen content decreased strongly in the intermediate stages
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Fig. 2.
13
C NMR spectra of the fulvic fraction extracted from the sludge/plant mixture during the composting process.
by 15%, towards the end of composting it increased, as did the O/C ratio. In the FTIR spectra, as composting progressed, there was a loss in absorbance around 1072 cm 1, with an increase in absorbance at around 1123–1140 cm 1 (Fig. 1). The 13C NMR analysis showed a roughly 37% decrease in the signal of N- or O-alkyls (around 62, 71 and 101 ppm), while there was a marked increase in the resonance of alkyl-C, aromatic-C and carboxyl-C (Fig. 2).
These structure changes in the fulvic fraction suggest that microbial communities developed when composting can take advantage of the availability of polysaccharides, readily broken down as an energy source (Senesi et al., 1996; Hafidi et al., 2005b). Other aliphatic compounds, which mainly show absorbance around 1140 cm 1, and resonance in the C-alkyl area around 33 ppm, seem to be neoformed or preserved. There were also ether/ester bonds among the aliphatic carbons preserved. In fact a strong
A. Jouraiphy et al. / Bioresource Technology 99 (2008) 1066–1072 Table 3 Proportions of aliphatic, aromatic and carboxylic carbon in fulvic fraction from different stages of the composting process Samples (days)
C-alkyla
O-alkyl/N-alkyla
C-aromatica
C-carboxylica
Raw mixture 60 days 135 days
15.03 24.01 23.72
55.77 37.00 33.82
9.53 8.67 17.52
19.65 30.31 24.92
a
Percentages obtained from ratio of integrated areas of the spectrum to the whole spectrum area.
resonance signal was recorded around 168 and 174 ppm towards the end of composting. Similar results have been found in a previous study of fulvic structures in the course of the composting of mixtures of lagooning sludge and straw (Amir et al., 2005). In this study, the authors suggest that the accumulation of relatively resistant aliphatic polyesters/ethers could contribute to the formation of fulvic structures during composting. Inbar et al. (1991) reported in FTIR and NMR studies of grape marc during composting, the relative intensity of the main peaks in the aliphatic region (1100–950 cm 1; 75 and 108 ppm), which are attributed to carbohydrates and/or aliphatic alcohols. Their intensity decreased slightly, while the peak at 173 ppm, resulting from COOH and ester groups, increased in intensity during the composting process. Numerous authors, have reported the production of stable aliphatic structures rich in carboxyl groups from microbial activities (Dudley et al., 1990; Zhang et al., 1993; Lehtonen et al., 2001). Lorenz et al. (2000) reported that during humification, the sharp peak at 33 ppm comes from the accumulation of long-chain CH2 from cutin, suberin and plant waxes although residues of microbial biomass may also contribute to this region. Lehtonen et al. (2001) strongly support the suggestion that the highly aliphatic macromolecular biolipids represent potential substrates for intra-molecular n-alkane production within the humic acid structure due to the presence of hydroxy and dicarboxylic fatty acids that potentially facilitate esterification of carboxylic acid and hydroxyl groups in the humic molecules. Schnitzer et al. (2000) found an increase in molecular bonds and inter- and intra-molecular associations of the organic matter during the composting process. This indicates the formation of large molecules which are chemically and biologically less reactive than the initial matter from which they are derived. Therefore, these aliphatic polyesters/ethers can be considered as new structures incorporated into the fulvic fraction during the humification process. Moreover, in the final compost (135 days), the intensity of the aromatic structures increased to reach twice the levels in the initial mixture (Table 3). Indeed, the increase of aromaticity has also been demonstrated to be an index of humification in the course of composting (Senesi et al., 1996; Castaldi et al., 2005). The same was found by Amir et al. (2005) in a structural study of the fulvic fraction dur-
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ing the composting of a mixture of lagooning sludge and straw. During the maturation phase, there was a considerable increase in aromatic compounds leading to intense peaks at 3400 and 1640 cm 1 as well as an increase in the resonance at 120–168 ppm. Likewise, Gigliotti et al. (2001) found that fulvic structures from composted sludge can be characterized by their aromatic properties and by the presence of carboxyl and amide groups, thus having a much more complex molecular structure than soil fulvic acids. In agreement with the works of Chen et al. (1989), Inbar et al. (1991) also reported a decrease in carbohydrate compounds, accompanied by an increase in aromaticity and carboxyl content. These trends in the course of composting suggest that microbial metabolization starts by breaking down the carbohydrates such as polysaccharides. At the same time, fulvic structures incorporated more aromatic and stable aliphatic structures that are chemically and biologically less reactive than the starting material. 4. Conclusions The fulvic fraction extracted from activated sludge mixed with plant matter is mainly composed of polysaccharides, polypeptides and aromatic structures. During the composting process, the breakdown of organic matter and subsequent humification involves a decrease of polysaccharide content and the production of structures incorporating more aromatic compounds and aliphatic polyesters/ethers. It has been suggested that the latter are neoformed with an increase in ether/ester bonds leading to the formation of alkyl linkages during the humification. Thus, the final product presents both aromatic and aliphatic structures that are chemically and biologically more stable than the initial material. Therefore, monitoring the structure of the fulvic fraction could provide information on the humification process and the maturity of the compost. References Ait Baddi, G., Hafidi, M., Cegarra, J., Alburquerque, J.A., Gonza´lvez, J., Gilard, V., Revel, J.-C., 2004. Characterization of fulvic acids by elemental and spectroscopic (FTIR and 13C-NMR) analyses during composting of olive mill wastes plus straw. Bioresour. Technol. 93, 217–232. Amir, S., Hafidi, M., Lemee, L., Bailly, J-R., Merlina, G., Guiresse, M., Pinelli, E., Revel, J.-C., Amble`s, A., 2006. Structural characterization of humic acids, extracted from sewage sludge during composting, by Thermochemolysis–Gas Chromatography–Mass Spectrometry. Process Biochem. 41 (2), 410–422. Amir, S., 2005. Contribution a` la valorisation des boues de stations d’e´puration par compostage: devenir des micropolluants me´talliques et organiques et bilan humiques du compostage. Ph.D. Universite´ Cadi Ayad Marrakech. Amir, S., Hafidi, M., Merlina, G., Revel, J.-C., 2005. Structural characterization of fulvic acids during composting of sewage sludge. Process Biochem. 40 (5), 1693–1700. Castaldi, P., Alberti, G., Merella, R., Melis, P., 2005. Study of the organic matter evolution during municipal solid waste composting aimed at
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identifying suitable parameters for the evaluation of compost maturity. Waste Manage. 25, 209–213. Chen, Y., Inbar, Y., Hadar, Y., Malcolm, R.L., 1989. Chemical properties and solid-state CPMAS 13C-NMR of composted organic matter. Sci. Total Environ., 201–208. Da Silva, J.C.G.E., Machado, A.A.S.C., Silva, M.A.B.A., 1998. Acid-base properties of fulvic acids extracted from an untreated sewage sludge and from composted sludge. Water Res. 32 (2), 441–449. Doane, T.A., Deveˆvre, O.C., Horwa`th, W.R., 2003. Short-term soil carbon dynamics of humic fractions in low-input and organic cropping systems. Geoderma 114, 319–331. Dudley, R.L., Fyfe, C.A., Preston, C.M., 1990. A P13C-CPMAS NMR spectroscopic study of the transformation of plant material to peat and coal. Can. J. Spectrosc. 35, 31–35. Gigliotti, G., Businelli, D., Giusquiani, P.L., 2001. Composition changes of soil humus after massive application of urban waste compost: a comparison between FTIR spectroscopy and humification parameters. Agronomie 21, 169–178. Hafidi, M., Ait Baddi, G., Gilard, V., Ramos, P., Alimi, K., Revel, J.-C., 2005a. Thermal and spectroscopic characterization of fulvic acids during olive mill waste composting. Chem. Ecol. 21 (4), 313–324. Hafidi, M., Amir, S., Revel, J.-C., 2005b. Structural characterization of olive mill wastewater after aerobic digestion using elemental analysis, FTIR and 13C-NMR. Process Biochem. 40 (8), 2615–2622. Hayes, M.H.B., MacCarthy, P., Malcolm, R.L., 1989. In: Swift, R.S. (Ed.), Humic Substances II—In Search of Structure. Wiley, Chichester. Inbar, Y., Chen, Y., Hadar, Y., 1991. Carbon-13 CPMAS NMR and FTIR spectroscopic analysis of organic matter transformations during composting of solid wastes from Wineries. J. Soil Sci. 152, 272–282. Inbar, Y., Hadar, Y., Chen, Y., 1992. Characterization of humic substances formed during the composting of solid wastes from wineries. Sci. Total Environ. 113, 35–48. Jarde, E., Mansuy, L., Faure, P., 2003. Characterization of the macromolecular organic content of sewage sludges by thermally assisted hydrolysis and methylation–gas chromatography–mass spectrometery (THM–GC/MS). J. Anal. Appl. Pyrol. 331–350. Joaquim, C.G., Esteves, D.S.A., Diklio, A.S., Machado, C., Monica, A.B.A.S., 1998. Acid-base properties of fulvic acids extracted from an untreated sewage sludge and from composted sludge. Water Res. 32, 441–449. Jouraiphy, A., Amir, S., ElGharous, M., Revel, J.C., Hafidi, M., 2005. Chemical and spectroscopic analysis of organic matter transformation during composting of sewage sludge and green plant waste. Int. Biodeterior. Biodegrad. 56, 101–108. Kavira, J., Sharma, S., 2003. Municipal solid waste management through vermicomposting employing exotic and local species of earthworms. Bioresour. Technol. 90, 169–173. Lehtonen, K., Ha¨nninen, K., Ketola, M., 2001. Structurally bound lipid in peat humic acids. Org. Geochem. 32, 33–43. Lguirati, A., Ait Baddi, G., El Mousadik, A., Gilard, V., Revel, J.C., Hafidi, M., 2005. Structural characteristics of humic acids extracted from urban waste landfill mould. Int. Biodeterior. Biodegrad. 56, 8–16. Lichtfouse, E., Chenu, C., Baudin, F., Leblond, C., DaSilva, M., Behar, F., Derenne, S., Largeau, C., Wehrung, P., Albrecht, P., 1998. A novel
pathway of soil organic matter formation by selective preservation of resistant straight-chain biopolymers: chemical and isotope evidence. Org. Geochem. 28, 411–415. Lorenz, K., Preston, C.M., Raspe, S., Morrison, I.K., Feger, K.H., 2000. Litter decomposition and humus characteristics in Canadian and German spruce ecosystems: information from tannin analysis and 13 C-CPMAS NMR. Soil Biol. Biochem. 32, 779–792. Malcolm, R.L., 1990. Humic Substances in Soil and Crop Sciences, vol. 677. American Society of Agronomy and Soil Science Society of America, Madison, USA. Mathur, S.P., Dowen, G., Dinel, H., Schnitzer, M., 1993. Determination of compost biomaturity. I. Literature review. Biol. Agri. Horticul. 10, 65–85. McDonnell, R., Holden, N.M., Ward, S.M., Collins, J.F., Farrell, E.P., Hayes, M.H.B., 2001. Characteristics of humic substances in heathland and forested peat soils of the Wicklow Mountains. Bio. Environ. 101B (3), 187–197. Ouatmane, A., 2000. Etude du compostage de quelques de´chets organiques: I – Approche physico-chimique, calorimetriques et spectroscopique de l’e´tude de l’e´tat de maturation des composts. II-Analyse qualitative et quantitative de la fraction humique, Ph.D., Faculte´ de Sciences Semlalia, Marrakech, Maroc, 170p. Ouatmane, A., Provenzano, M.R., Hafidi, M., Senesi, N., 2000. Compost maturity assessment using calorimetry, spectroscopy and chemical analysis. Comp. Sci. Util. 8 (2), 135–146. Piccolo, A., 2002. The supramolecular structure of humic substances: a novel understanding of humus chemistry and implications in soil science. Adv. Agron. 75, 57–134. Piccolo, A., Zaccheo, P., Genevini, P.G., 1992. Chemical characterization of humic substances extracted from organic-waste-amended soils. Bioresour. Technol. 40, 275–282. Schnitzer, M., 1978. Humic substances: chemistry and reactions. In: Schnitzer, M., Khan, S.U. (Eds.), Soil Organic Matter. Elseiver, Amsterdam, pp. 1–64. Schnitzer, M., Dinel, H., Schulten, H.R., Pare´, T., Lafond, S., 2000. Humification of duck farm wastes. In: Ghabour, E.A., Davies, G. (Eds.), Humic Substances: Versatile Components of Plants, Soil and Water. The Royal Society of Chemistry, pp. 20–34. Senesi, N., Miano, T.M., Brunetti, G., 1996. Humic substances in organic amendments and effects on native soil humic substances. In: Piccolo, A. (Eds.), Humic Substances in Terrestrial Ecosystems, pp. 531–593. Wagner, W.C., 1999. Sustainable agriculture: how to sustain a production system in a changing environment. Int. J. Parasitol. 29, 1–5. Zbytniewski, R., Buszewski, B., 2005. Characterization of natural organic matter (NOM) derived from sewage sludge compost. Part 1: Chemical and spectroscopic properties. Bioresour. Technol. 96, 471–478. Zhang, E., Hatcher, P.G., Davies, A., 1993. Chemical composition of pseudo-phlobaphinite precursors: Implications for the presence of aliphatic biopolymers in vitrinite coal. Org. Geochem. 20, 721–734. Zorpas, A.A., Arapoglou, D., Panagiotis, K., 2003. Waste paper and clinoptilolite as a bulking material with dewatered anaerobically stabilized primary sewage sludge (DASPSS) for compost production. Waste Manage. 23, 27–35.
Bioresource Technology 99 (2008) 1066–1072
Structural study of the fulvic fraction during composting of activated sludge–plant matter: Elemental analysis, FTIR and 13C NMR A. Jouraiphy a, S. Amir c, P. Winterton e, M. El Gharous a, J-C. Revel d, M. Hafidi
b,*
a Labo. Fertilite´ des Sols, Centre Arido-Culture, INRA Settat, Morocco Equipe Ecologie Ve´ge´tale, Sol et Environnement, Faculte´ des Sciences Semlalia, De´partement de Biologie, BP/2390 Marrakech, Morocco c De´partement de Biologie, Faculte´ Polydisciplinaire, Beni Mellal, Morocco Equipe Agronomie, Environnement et Ecotoxicologie (A2E), Ecole Nationale Supe´rieure Agronomique, Auzeville-Tolosane, BP/107 Toulouse, France e Universite´ Paul Sabatier, Toulouse, France b
d
Received 25 April 2006; received in revised form 26 February 2007; accepted 27 February 2007 Available online 18 April 2007
Abstract The starting fulvic structures isolated from an initial mixture of activated sludge and plant matter presented abundant peptide structures and hydrocarbons that absorb in FTIR spectra around (1650 and 1560 cm 1) and 1072 cm 1, respectively. They also present a high resonance signal in the O- and N-alkyl areas of 13C NMR spectra. As composting proceeded, some changes led to the formation of the molecular structures of fulvic fraction as demonstrated by a decrease of intensity of compounds absorbing around 1072 cm 1 and an increase of those absorbing around 1140 cm 1. The resonance of O- and N-substituted alkyl carbon also decreased from 55.7% to 33.8%, with an increase in the intensity of aromatic carbons, alkyls and carboxyls. These data indicate that the microbial community that developed during composting used polysaccharides as an energy source, structures which are supplied in abundance in the initial material. The fulvic fraction of the final compost is much richer in aromatic structures and aliphatic ethers/esters, which are most likely preserved from the original material but probably also synthesized through the microbial activities. The occurrence of alkyl ethers/esters at the end of composting is demonstrated by strong absorbance around 1140 cm 1 in the FTIR spectra and large peaks at 32 and 174 ppm in the NMR spectra. These structures could also be produced following the creation of ether/ester bonds during the humification process. 2007 Elsevier Ltd. All rights reserved. Keywords: Fulvic fraction; Compost; Polysaccharides; Resistant aliphatic; Aromaticity
1. Introduction For several decades, the increases in agricultural production have been closely linked to the use of chemical fertilizers (Wagner, 1999; Ouatmane, 2000). These fertilizers, however beneficial to productivity, contribute to the pollution of food chains and of water tables. To compensate for these risks and with a view to enriching soil impoverished by erosion and the intensification of farming, interest in
*
Corresponding author. E-mail address: hafi[email protected] (M. Hafidi).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.02.031
recycling organic waste has gained momentum (Kavira and Sharma, 2003). Several techniques are proposed in the scientific literature to deal with this waste (e.g. incineration, controlled tipping). However, most of these techniques are costly, require specialist personnel and have limitations (Amir, 2005; Lguirati et al., 2005). Therefore, composting is a more accessible technique for recycling organic waste and transforming it into an ecologically stable product agronomically beneficial for the soil (Ouatmane et al., 2000; Jouraiphy et al., 2005). However, the success of the composting process is gauged by the quality of the final product, especially its stability. In this context, numerous studies have been carried out to guarantee the
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stability and maturity of composts, using several different ways to approach the problem: observation of empirical evidence, physicochemical or microbiological studies, spectroscopy or humic examination (Mathur et al., 1993; Amir, 2005). Some works (Inbar et al., 1992) have revealed that the maturity of compost is closely linked to the levels of humic substances formed during the final stages of the composting process. However, the heterogeneity of these substances presents a serious obstacle to the determination of their exact chemical structure as well as to learning about the processes involved in their formation (Hayes et al., 1989). In general, humic substances are composed of a very large variety of aromatic rings (phenolic and quinonic) bound by acid functional groups or by peripheral aliphatic chains (polysaccharides, peptides, etc.) and grouped into the different molecular arrangements that make up the structure of humic substances (Piccolo, 2002; Jarde et al., 2003; Amir et al., 2006). These can be divided mainly into humic and fulvic fractions). Despite the quantity of works on the subject of humic substances, information about the fulvic fraction remains limited, probably because of their chemical complexity (Da Silva et al., 1998; Joaquim et al., 1998; Ait Baddi et al., 2004; Hafidi et al., 2005a). The objective of this study is to gain a better understanding of the humification process and the formation of fulvic structures throughout the different stages of composting of activated sludge. Various complementary techniques, such as elemental analysis, Fourier transform infra-red spectroscopy (FTIR) and carbon 13 Nuclear Magnetic Resonance (13C NMR) were all used at the different stages of the composting process. 2. Methods
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to be distinguished. The stabilization phase where the average temperature of the heap raises to 72 C within a matter of days. The maturation phase presenting a steady fall in temperature as it slowly reaches equilibrium with ambient temperature (Jouraiphy et al., 2005). Samples of compost were taken at different stages of the process from raw mixture, after 30, 60 and 135 days. The sampling was done at each stage from different places within the heap (center, surface, core of the heap, sides) and after thorough mixing of the various sub-samples, a representative 500 g portion was collected and refrigerated until analysis. Table 1 shows the variations of certain physicochemical properties of the mixture at different stages of the composting process. 2.2. Extraction of fulvic fraction The fulvic fraction was extracted from a fresh 30 g sample. It was defatted using 120 ml of a chloroform–methanol solvent mixture (2:1) (Lichtfouse et al., 1998). This step was repeated three times at 4 C. The solvent was then evaporated off and the samples were treated three times with 40 ml of distilled water so as to extract the water-soluble non-humic substances (sugars, proteins). Then, the humic substances were extracted with 40 ml 0.1 M NaOH. This was repeated several times until the extract obtained was colourless. After filtration, the solutions were pooled and the humic acids were then precipitated out of solution with 1.5 M H2SO4 for 24 h at 4 C. The solution containing the fulvic fraction was dialyzed with a Spectra-Por membrane (1000 Da) to eliminate excess salts. The content of fulvic structures was then calculated after freeze drying. 2.3. Elemental analysis
2.1. Composting The activated sludge was taken from the aerobic waste water treatment plant of Khouribga (Morocco). Dried sludge (10 m3) was mixed with 5 m3 of fresh plant matter and left in a prism-shaped heap (L = 8 m · h = 1.5 m) on a composting platform. The mixture was prepared so as to have the optimal parameters for good composting, namely 60% humidity and a C/N ratio of around 30. The heap was turned every fortnight to maintain good aerobic conditions and was watered. The temperature of the compost heap was checked every day in several locations. The temperature data enables two successive phases
Elemental analysis was carried out using a Carbograph Autoanalyser (Fison Carlo Erba EA 1110) to analyse C, H and N and a type EA 1106 instrument to analyse the O of the humic acids. 2.4. Fourier transform infra-red spectroscopy Two mg of fulvic fraction extracted from each compost sample were mixed with 250 mg of KBr. The absorption spectra of the pellets were recorded on a Perkin Elmer 1600 FTIR spectrophotometer from 4000 to 400 cm 1 at 16 nm/s.
Table 1 Variation of the physico-chemical characteristics of composted sewage sludge and plant matter Stage
pH
C/N
Dec%a
Humic fraction (g kg
Raw mixture 30 days 60 days 135 days
6.36 ± 0.24 5.7 ± 0.18 6.1 ± 0.33 6.23 ± 0.12
29.75 25.98 23.76 12.15
– 6.6 ± 0.33 29.3 ± 0.58 60.8 ± 0.68
26.5 ± 1.55 30.0 ± 1.39 39.7 ± 0.47 39.5 ± 0.27
a
% Dry weight; Dec%: % decomposition; HA: humic acids.
1
dry matter)
Fulvic fraction (g kg 38.2 ± 0.97 22.2 ± 1.46 11.4 ± 2.4 17.8 ± 1.3
1
dry matter)
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2.5. Carbon-13 nuclear magnetic resonance The samples were prepared by dissolving 100 mg of fulvic fraction in 3 ml of NaOD/D2O (0.5 M). The 13C NMR spectrum was recorded with a Bruker ES-WB 300 spectrometer working at 75.469 MHz. A gated pulse decoupling technique was used to suppress any increase in signal intensity due to the Nuclear Overhauser effect. The spectrum was recorded in the following conditions: relaxation: 1.8 s, acquisition period: 0.98 s, the pulse: 35. Total acquisition required 72 h. The areas of the spectra that were integrated were 0–55 ppm (aliphatic carbon), 55– 110 ppm (aliphatic carbon substituted with O or N), 110– 165 ppm (aromatic carbon), and 165–200 ppm (carboxylic carbon). 3. Results and discussion During the stabilisation phase of composting of activated sludge and plant matter, the presence of easily broken down substances allows intense microbial activity, which raises the average temperature of the heap to 72 C in the first days of composting. Secondly comes the maturation phase where temperature decreases because the remaining organic matter (mainly lignocellulose compounds) resists microbiological attack. The C/N ratio and the degree of decomposition reached 12% and 60% respectively after 135 days of composting the activated sludge–plant matter mixture (Table 1). The results illustrate successful composting: similar results have been found in other studies (Zorpas et al., 2003; Zbytniewski and Buszewski, 2005). Compost maturity has also been indicated by the increase in the level of humic acids within the compost (Table 1). Numerous authors have defined the maturity of composts by their level of humic substances: ‘‘humic and fulvic fractions’’. However, in this work, the level of fulvic fraction decreased in the course of composting (Table 1). Some authors suggest that fulvic fraction degradation products are precursors for the formation of humic acids (Doane et al., 2003). Generally, the microbial communities that develop during the composting acquire their energy from degradation of the substances most easily broken down and this could include the compounds that make up the fulvic structures. The biooxidation of these compounds leads to the production of substances with high levels of stable structures, which indicate compost maturity.
Hence the importance of studying in greater depth the evolution of fulvic structures in the course of composting. The correlations between the data gained from the three techniques used: elemental analysis; FTIR; 13C NMR have been commonly investigated to determine the structures composing the humic substances and their evolution through the advancement of the humification process in the course of composting (McDonnell et al., 2001). See Table 2 for the elemental composition and atomic ratios of the fulvic fraction extracted from the compost of activated sludge and plant matter at the various stages of composting. The structures of the fulvic fraction isolated from the uncomposted starting material, compared to the elemental composition of soil (Schnitzer, 1978), show their high content in O, N and O/C ratio; but a low C/H and C/N ratios of about 0.5 and 9.5, respectively. This indicates their richness in aliphatic structures with high O and N contents. Senesi et al. (1996), based on the review of numerous studies of FA in sludge, suggest that lower C/N and C/H ratios may be attributed to the high content of protein decomposition products and aliphatic components respectively in sludge fulvic fraction. In fact, the FTIR spectra of raw mixtures of activated sludge and plant matter exhibit high absorbance at 1650 and 1560 cm 1, attributed to aromatics and peptides, and around 1124 and 1072 cm 1 assigned to the aliphatic structures with high levels of functional groups rich in oxygen (Fig. 1). Similar results were found by Malcolm (1990) in the IR spectra of fulvic acids extracted from sludge, characterized by the presence of aliphatic and aromatic compounds as well as by the breakdown products of the proteins and polysaccharides. The 13C NMR spectra of raw mixture also exhibited strong resonance of oxygen- or nitrogen-substituted alkyl carbons that present amides at 62 ppm, and a strong signal around 71.29 ppm assigned to hydrocabons and anomeric carbon at 101.11 ppm, and many functional entities such as hydroxyls (168.86 ppm) and carboxyls (174.87 ppm) (Fig. 2). Calculations of the areas under the curves of the regions of aliphatic, aromatic and carboxylic carbons are shown in Table 3. The resonance signal in O- and N-alkyl areas presents about of 55.7%. Piccolo et al. (1992) attributed these nitrogen and oxygen-rich compounds to the high levels of non-humified biomolecules (polysaccharides and polypeptides).
Table 2 Elemental composition and atomic ratios of the fulvic fraction at different stages of the composting process of the sludge/fresh plant waste mixture Stages of the composting process
Raw mixture 30 days 60 days 135 days Fulvic acids from soila a
C (%)
35.10 34.61 33.47 31.21 45.70
Fulvic acid of soil model (Schnitzer, 1978).
N (%)
4.33 4.28 4.15 4.21 2.10
H (%)
5.50 5.21 5.26 4.55 5.40
O (%)
44.81 39.00 38.00 44.30 44.80
Ash (%)
10.26 16.90 19.12 15.73 –
Atomic ratios C/N
C/H
O/C
9.46 9.43 9.41 8.65 25.50
0.53 0.55 0.53 0.57 0.70
0.96 0.85 0.85 1.06 0.70
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Fig. 1. FTIR spectra of the fulvic fraction extracted from composted activated sludge during the composting process.
At the end of the composting period, the carbon and hydrogen content of the fulvic fraction diminished by about 11% and 17% of the initial amount, respectively
(Table 2). No notable changes were observed in the N content, but the atomic ratio C/N decreased. While, the oxygen content decreased strongly in the intermediate stages
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Fig. 2.
13
C NMR spectra of the fulvic fraction extracted from the sludge/plant mixture during the composting process.
by 15%, towards the end of composting it increased, as did the O/C ratio. In the FTIR spectra, as composting progressed, there was a loss in absorbance around 1072 cm 1, with an increase in absorbance at around 1123–1140 cm 1 (Fig. 1). The 13C NMR analysis showed a roughly 37% decrease in the signal of N- or O-alkyls (around 62, 71 and 101 ppm), while there was a marked increase in the resonance of alkyl-C, aromatic-C and carboxyl-C (Fig. 2).
These structure changes in the fulvic fraction suggest that microbial communities developed when composting can take advantage of the availability of polysaccharides, readily broken down as an energy source (Senesi et al., 1996; Hafidi et al., 2005b). Other aliphatic compounds, which mainly show absorbance around 1140 cm 1, and resonance in the C-alkyl area around 33 ppm, seem to be neoformed or preserved. There were also ether/ester bonds among the aliphatic carbons preserved. In fact a strong
A. Jouraiphy et al. / Bioresource Technology 99 (2008) 1066–1072 Table 3 Proportions of aliphatic, aromatic and carboxylic carbon in fulvic fraction from different stages of the composting process Samples (days)
C-alkyla
O-alkyl/N-alkyla
C-aromatica
C-carboxylica
Raw mixture 60 days 135 days
15.03 24.01 23.72
55.77 37.00 33.82
9.53 8.67 17.52
19.65 30.31 24.92
a
Percentages obtained from ratio of integrated areas of the spectrum to the whole spectrum area.
resonance signal was recorded around 168 and 174 ppm towards the end of composting. Similar results have been found in a previous study of fulvic structures in the course of the composting of mixtures of lagooning sludge and straw (Amir et al., 2005). In this study, the authors suggest that the accumulation of relatively resistant aliphatic polyesters/ethers could contribute to the formation of fulvic structures during composting. Inbar et al. (1991) reported in FTIR and NMR studies of grape marc during composting, the relative intensity of the main peaks in the aliphatic region (1100–950 cm 1; 75 and 108 ppm), which are attributed to carbohydrates and/or aliphatic alcohols. Their intensity decreased slightly, while the peak at 173 ppm, resulting from COOH and ester groups, increased in intensity during the composting process. Numerous authors, have reported the production of stable aliphatic structures rich in carboxyl groups from microbial activities (Dudley et al., 1990; Zhang et al., 1993; Lehtonen et al., 2001). Lorenz et al. (2000) reported that during humification, the sharp peak at 33 ppm comes from the accumulation of long-chain CH2 from cutin, suberin and plant waxes although residues of microbial biomass may also contribute to this region. Lehtonen et al. (2001) strongly support the suggestion that the highly aliphatic macromolecular biolipids represent potential substrates for intra-molecular n-alkane production within the humic acid structure due to the presence of hydroxy and dicarboxylic fatty acids that potentially facilitate esterification of carboxylic acid and hydroxyl groups in the humic molecules. Schnitzer et al. (2000) found an increase in molecular bonds and inter- and intra-molecular associations of the organic matter during the composting process. This indicates the formation of large molecules which are chemically and biologically less reactive than the initial matter from which they are derived. Therefore, these aliphatic polyesters/ethers can be considered as new structures incorporated into the fulvic fraction during the humification process. Moreover, in the final compost (135 days), the intensity of the aromatic structures increased to reach twice the levels in the initial mixture (Table 3). Indeed, the increase of aromaticity has also been demonstrated to be an index of humification in the course of composting (Senesi et al., 1996; Castaldi et al., 2005). The same was found by Amir et al. (2005) in a structural study of the fulvic fraction dur-
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ing the composting of a mixture of lagooning sludge and straw. During the maturation phase, there was a considerable increase in aromatic compounds leading to intense peaks at 3400 and 1640 cm 1 as well as an increase in the resonance at 120–168 ppm. Likewise, Gigliotti et al. (2001) found that fulvic structures from composted sludge can be characterized by their aromatic properties and by the presence of carboxyl and amide groups, thus having a much more complex molecular structure than soil fulvic acids. In agreement with the works of Chen et al. (1989), Inbar et al. (1991) also reported a decrease in carbohydrate compounds, accompanied by an increase in aromaticity and carboxyl content. These trends in the course of composting suggest that microbial metabolization starts by breaking down the carbohydrates such as polysaccharides. At the same time, fulvic structures incorporated more aromatic and stable aliphatic structures that are chemically and biologically less reactive than the starting material. 4. Conclusions The fulvic fraction extracted from activated sludge mixed with plant matter is mainly composed of polysaccharides, polypeptides and aromatic structures. During the composting process, the breakdown of organic matter and subsequent humification involves a decrease of polysaccharide content and the production of structures incorporating more aromatic compounds and aliphatic polyesters/ethers. It has been suggested that the latter are neoformed with an increase in ether/ester bonds leading to the formation of alkyl linkages during the humification. Thus, the final product presents both aromatic and aliphatic structures that are chemically and biologically more stable than the initial material. Therefore, monitoring the structure of the fulvic fraction could provide information on the humification process and the maturity of the compost. References Ait Baddi, G., Hafidi, M., Cegarra, J., Alburquerque, J.A., Gonza´lvez, J., Gilard, V., Revel, J.-C., 2004. Characterization of fulvic acids by elemental and spectroscopic (FTIR and 13C-NMR) analyses during composting of olive mill wastes plus straw. Bioresour. Technol. 93, 217–232. Amir, S., Hafidi, M., Lemee, L., Bailly, J-R., Merlina, G., Guiresse, M., Pinelli, E., Revel, J.-C., Amble`s, A., 2006. Structural characterization of humic acids, extracted from sewage sludge during composting, by Thermochemolysis–Gas Chromatography–Mass Spectrometry. Process Biochem. 41 (2), 410–422. Amir, S., 2005. Contribution a` la valorisation des boues de stations d’e´puration par compostage: devenir des micropolluants me´talliques et organiques et bilan humiques du compostage. Ph.D. Universite´ Cadi Ayad Marrakech. Amir, S., Hafidi, M., Merlina, G., Revel, J.-C., 2005. Structural characterization of fulvic acids during composting of sewage sludge. Process Biochem. 40 (5), 1693–1700. Castaldi, P., Alberti, G., Merella, R., Melis, P., 2005. Study of the organic matter evolution during municipal solid waste composting aimed at
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