Electrofocusing the compost organic matter obtained by coupling SEC–PAGE

Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 4360–4367

Electrofocusing the compost organic matter obtained by coupling SEC–PAGE Luciano Cavani

b

a,*

, Olga Trubetskaya b, Marco Grigatti a, Oleg Trubetskoj c, Claudio Ciavatta a

a Dipartimento di Scienze e Tecnologie Agroambientali, Alma Mater Studiorum, Universita` di Bologna, Viale Fanin 40, I-40127 Bologna, Italy Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia c Institute of Basic Biological Problems, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia

Received 28 June 2006; received in revised form 4 June 2007; accepted 23 August 2007 Available online 23 October 2007

Abstract Humic acids (HA)-like extracted from compost at the beginning (t0) and after 130 days of composting (t130) were fractionated by coupling size exclusion chromatography to polyacrylamide gel electrophoresis (SEC–PAGE). HA-like fractions with the same molecular size (MS) and electrophoretic mobility were pooled and further characterised by analytical polyacrylamide gel electrofocusing (EF) and compared with HA separated from a Typic Chernozem soil. During the composting process all fractions were subjected to quantitative and qualitative modifications: the high MS fraction was degraded, the mid MS fractions were qualitatively changed, the content of low MS fractions increased and changed qualitatively. The main changes in EF pattern of the non fractionated HA-like t130 were associated to low MS fractions. Such data seem to be reliable for explanation what mechanisms and monitoring of the evolution of the compost organic matter for their agricultural uses.  2007 Elsevier Ltd. All rights reserved. Keywords: Compost; Electrofocusing; Humic substances; Humic acids-like; SEC–PAGE

1. Introduction The recycling of composted organic wastes provides the possibility to supply nutrients and large quantities of humic substances (HS) to soil for improving soil physico-chemical and biological properties (Senesi and Brunetti, 1996) and to enhance the level of organic carbon which become an effective and low cost carbon sink to reduce gas emissions and greenhouse effect (Marmo, 2000). Compost HS are formed within a shorter time period than the soil HS, therefore it is important to understand the chemical and physical transformations of the compost HS. In practice, the compost quality is evaluated by simple chemical parameters, as C/N ratio, degree of humification (Ciavatta and Govi, 1993), dissolved organic matter *

Corresponding author. Tel.: +39 051 2096209; fax: +39 051 2096203. E-mail address: [email protected] (L. Cavani).

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.08.078

(Zmora-Nahum et al., 2005), organic matter evolution index (Adani et al., 1997); or respirometric techniques, as oxygen uptake rate (Adani et al., 2001; Barrena Go´mez et al., 2005). However, these approaches gives us the possibility to obtain only an apparent evaluation of the humification process, but makes impossible to understand the ways and the mechanisms of formation of the HS during composting. Therefore, complex of methods are required to elucidate structural peculiarities of compost HS in a more sophisticated way, which allows qualitative and quantitative characterization of the end compost products to be made. Many different analytical procedures have been adopted as elemental and functional group analysis, fluorescence, IR, 1H and 13C NMR spectroscopy, thermochemolysisGC etc. (i.e. Chen and Inbar, 1993; Amir et al., 2006; Senesi et al., 2007), but these techniques have practical difficulties

L. Cavani et al. / Bioresource Technology 99 (2008) 4360–4367

and are not suitable to be applied to normal compost analytical investigations. It is also generally accepted today to consider HS as a kind of complex colloidal system with no exactly definable molecular size (MS) (Piccolo, 2001) and one of the physical–chemical parameters, which has been suggested for quality evaluation of HS formed during composting is MS distribution. Thus, size exclusion chromatography (SEC) has been extensively employed as rapid and versatile techniques enabling preparative quantities of HS fractions with different MS (Roletto et al., 1985; Fortun and Duato, 1986; Senesi and Brunetti, 1996; Trubetskaya et al., 2001; Brunetti et al., 2005). However, one of the main problems that often arise during SEC fractionation is interaction between HS and gel matrix, mainly through hydrogen bonds (De Nobili and Chen, 1999). The analytical isoelectrofocusing (IEF), an electrophoresis carried out in a pH gradient, has been used to study the HS extracted from the soils, sewage sludge and composts (Ceccanti et al., 1980; Tsiplenkov, 1983; De Nobili et al., 1989, 1990; Garcia et al., 1992; Ciavatta et al., 1993a, 1993b; Govi et al., 1993, 1994; Requena et al., 1996; Canali et al., 1998; Alianiello, 2003). The IEF fractionates the amphoteric compounds on the basis on their isoelectric point (pI), but in the case of HS the separation is principally controlled by the acidic groups and the separated bands depend on the pKa rather than the pI (Duxbury, 1989). Therefore, with HS the term electrofocusing (EF) is suggested (De Nobili, 1988; Duxbury, 1989), because the pH of the separated bands is only the ‘‘apparent’’ pI. The main questionable point of this technique is the interpretation of EF fingerprinting pattern. The main differences in EF fingerprinting patterns for compost HS before and after composting process, which have been found by the most of the investigators, were the appearance of several additional bands focused at pH > 4.5 after a proper composting process (De Nobili et al., 1989; Garcia et al., 1992; Govi et al., 1993; Ciavatta et al., 1993a, 1993b). This fact has been interpreted as an increase of the molecular size (MS) of HS occurring during the composting (De Nobili et al., 1989; Ciavatta et al., 1993a, 1993b), in fact the reduction of the charge-to-mass ratio of the HS increase the ‘‘apparent’’ pI (or the pKa). On the other hand, Trubetskaya et al. (2001) have shown the increasing of the content of electrophoretic low MS fraction in compost humic acids (HA)-like during composting. Recently, Alianiello and Baroccio (2004), analysing MS fractions of HA from soil and peat obtained by ultrafiltration, observed that the dependence of EF profile on MS is not a general rule. Grigatti et al. (2006), analysing by principal component analysis (PCA) the EF fingerprinting pattern of HS extracted from three different compost, found that the variations in EF profile occurring at pH > 4.4 seems to be formed by the evolution of compounds initially present in the region at pH < 4.4. But, the presence of readily degradable organic carbon and the intense activity of microorganism produced

4361

an overestimation in the EF profile in the region at pH > 4.4 in the first composting period, depending on the characteristics of the raw materials used for compost production. Probably, if the bands at pH > 4.5 are not linked to an increase on MS, it could be due to the increase of chemical stability by further rearrangements of the molecular fragments to form less acidic organic compounds. Nevertheless, the EF is a potentially useful and powerful technique to study the composting process and to evaluate the stability of compost, but the interpretation of EF profiles is often subjective and contradictory, because the electrofocusing behaviour of HS are not well understood. The aim of this work was to characterise by EF the fractions obtained by SEC–PAGE from HS extracted from soil and compost samples for better understanding the formation way of the electrofocusing fingerprint pattern. 2. Methods 2.1. Soil and compost samples The soil sample was taken from the A horizon (10– 20 cm) of Typic Chernozem soil (Kursk region, Russia). The soil characteristics have been reported elsewhere (Trubetskoj et al., 1991; 1999). It should be noted that Chernozem soil is one of the most fertile soil, which engage the considerable part (central and south) of European and Asian section of Russia (Kononova, 1966) and Central Europe (Eckmeier et al., 2007). Moreover, the physicchemical parameters of Chernozem soil were similar to that of soil HA-IHSS standard 1S102 H (Eckmeier et al., 2007). Detailed characteristics of the compost samples used in this study have been reported earlier (Trubetskaya et al., 2001). Briefly, two compost samples, were composed by a mixture of 50% (w/w) of the sewage sludge and trimming, were analysed at the beginning (t0) and at the end of the composting process after 130 days (t130). The main physical-chemical characteristics of the compost samples are reported in Table 1. 2.2. Extraction of humic acids-like The HA-like were extracted from t0 and t130 compost samples, previously crushed and sieved to 0.25 mm sieve, using a solution of 0.5 M NaOH at a room temperature, under N2, for 6 h in a Dubnoff water-bath, according to Ciavatta and Govi (1993). The suspension was centrifuged at 5000g for 30 min and then filtered through a 0.22 lm filter using a Minitan S System (Millipore, Bedford MA – USA). The solution was acidified with 5 M HCl to pH < 2, let stand 1 h to precipitate HA-like and afterwards was centrifuged at 5000g for 20 min in order to eliminate the supernatant. The HA-like after suspension in HCl 10 3 M were dialysed against distilled water (d-H2O) until a neutral pH was achieved, then were freeze-dried and stored under vacuum over silica gel before use.

4362

L. Cavani et al. / Bioresource Technology 99 (2008) 4360–4367

Table 1 Physical–chemical characteristics of compost samples at the beginning (t0) and at the end (t130) of the composting process (data expressed to the dry matter) Parameter

t0

t130

Ash (%) pH (in water; 1:5 ratio w:v) Electrical conductivity (dS m 1) Total organic carbon (C, g kg 1) Total humic carbon (C, g kg 1) Dissolved organic carbon (C, g g 1) Total nitrogen (N, g kg 1) C/N ratio Total iron (Fe, g kg 1) Total potassium (K, g kg 1) Total phosphorus (P, g kg 1) Total sodium (Na, g kg 1) Total copper (Cu, mg kg 1) Total nickel (Ni, mg kg 1) Total chromium (Cr, mg kg 1) Total lead (Pb, mg kg 1) Total boron (B, mg kg 1) Total cadmium (Cd, mg kg 1)

37 7.4 2.5 297 34.8 16.3 222 13.4 18.6 10.1 5.1 1.2 146 46 35 35 32 3

48 7.7 2.0 220 50.4 6.2 155 14.2 18.2 6.9 6.1 1.8 141 44 53 30 34 3

2.3. Fractionation by tandem SEC–PAGE Fractionation of compost HA-like and Chernozem HA by tandem SEC–PAGE has been previously reported (Trubetskoj et al., 1997; 2001). Briefly: 10 mg HA was dissolved in 7 M urea and loaded onto a Sephadex G-75 (Pharmacia, Sweden) column (1.5 · 100 cm), equilibrated with the same solution. The void column volume (V0) and total column volume (Vt) were 42 and 158 ml, respectively. Column effluent was collected as 2 ml aliquots and each third aliquot was assayed by PAGE in the presence of denaturing agents according to Trubetskoj et al. (1991). The aliquots, forming individual electrophoretic zones in PAGE matrix with a similar EM in full, were combined into pools, dialysed against d-H2O, lyophilised, and used for further analysis. As elution volumes were similar for chromatographic pools, which formed bands with identical EM, it can be suggested that the MS of the fractions decreased in the sequence A > B > C + D, independently of the source of the HA. Based on ultrafiltration data the molecular weights 300–100, 100–30 and 30–5 kDa were found for fractions A, B and C + D, respectively (Trubetskoj et al., 1997, 2001). In order to obtain the preparative quantities of HA-like fractions the chromatographic procedure with subsequent electrophoretic analysis was repeated several times for HA-like t0 and HA-like t130 samples. The weight distribution of the HA-like fractions were calculated using the ratio Wi/RWi, where Wi is the weight of fraction and RWi the weight of all HA fractions obtained after SEC fractionation. 2.4. Electrofocusing of soil and compost samples The electrofocusing (EF) separation was carried out in 5% T [T = g (acrylamide + bis-acrylamide)/100 mL solu-

tion] and 3.33% C (C = g bisacrylamide/T) polyacrylamide slab gel (Bio-Rad, USA), containing 2% mixture of carrier ampholytes obtained blending Ampholine. (Pharmacia, Sweden) in the pH range 4.0–6.0 (70%) with (30%) Ampholine pH 3.5–10.0 (Ciavatta et al., 1993a). The slab (260 · 110 · 0.5 mm) was pre-run for 3 h in an electrophoretic cell (Multiphore II, LKB, Sweden), cooled at 2C and connected to a 2197 Power Supply (LKB, Sweden). The distance between the 2 electrodes was about 90 mm. The pre-set values were: 1200 V; 0.6 mA cm 1 and 0.9 W cm 1. After loading of samples (20 lL of NaOH 50 mM solution containing 5 mg mL 1 of freeze-dried sample) the run was carried out for 2 h. The pH gradient of the gel slab was immediately verified at the end of the run using a pH surface electrode (Ingold, Switzerland). The slab was stained for 2 h, under gentle continuous stirring, in a solution containing 15% glacial acetic acid (100%, Merck, Germany), 15% ethyl alcohol (95%, Carlo Erba, Italy), 0.25 g L 1 Coomassie Brilliant Blue R-250 (Merck, Germany) and 10 g L 1 copper (II) sulphate (99.5%, Carlo Erba, Italy) (first bath). The slab was then de-stained overnight, under a gentle continuous stirring, with a solution containing: 15% acetic acid, 15% ethyl alcohol, 0.025 g L 1 Coomassie Brilliant Blue R-250 and 10 g L 1 copper (II) sulphate (second bath) and finally with a solution prepared with 10% acetic acid and 10% ethyl alcohol (third bath) up to a complete destaining of the area without bands. After destaining, the focused bands were scanned at 633 nm with an Ultroscan XL Enhanced Laser Densitometer (LKB, Sweden). The bands of the EF densitograms were integrated and evaluated using the 2.1 version of the Gelscan Software (Pharmacia, Sweden). 3. Results and discussion 3.1. Coupling size exclusion chromatography– polyacrylamide gel electrophoresis (SEC–PAGE) PAGE in the presence of denaturing agents of the bulk HA-like samples is shown in Fig. 1. Each sample formed four naturally coloured fractions, named A, B, C and D with different electrophoretic mobility (EM): A – HA-like that did not move into the gel; B – a narrow band in the mid part of the gel; C and D – two bands at the bottom of the gel, these were combined into fraction C + D due to the relatively close electrophoretic behaviour. The intensity of all humic fractions increased considerably after composting (HA-like t130) in comparison with fresh compost, HA-like t0 (Fig. 1). In order to obtain preparative quantities of fractions A, B and C + D both samples were fractionated several time according to Trubetskoj et al. (1997). The percent weight composition with unfractionated HA-like respect to different fractions is shown in Table 2. The larger weight increase was observed for the fractions with low MS: fraction C + D increased 4 times, and mixture of the fraction BCD increased from 25 to 43%. It

L. Cavani et al. / Bioresource Technology 99 (2008) 4360–4367

Fig. 1. Electrophoresis of 0.15 mg HA-like t0 and HA-like t130 in 10% polyacrylamide gel in the presence of denaturing agents. The letters A, B, and C + D indicate discrete naturally coloured zones.

Table 2 Weight distribution (%) of the compost HA-like fractions obtained by coupling SEC–PAGE Samples

Fraction A Fraction B Fraction BCD Fraction C + D

HA-like t0 51 HA-like t130 27

19 17

25 43

3 14

seems that, during composting, the high MS fractions are transformed in the low MS fractions. 3.2. Electrofocusing (EF) 3.2.1. HS from soil Firstly, for a better understanding the electrofocusing behaviour of a HA fractions obtained coupling SEC– PAGE, the EF experiments were carried out on HA extracted from Chernozem soil (Fig. 2). The whole HA shows a EF pattern characterised by several bands focused in the 3.5–5.7 pH region (Fig. 2). The fraction A do not moves throughout the gel and does not shows any focused band in to the pH region analysed (approx. 3.5–8.0). This fraction did not move into the 10% polyacrylamide gel (Fig. 1) and during the SEC on Sephadex G-75 eluted in the exclusion volume. Many authors (De Nobili, 1988; Duxbury, 1989; Ciavatta and Govi, 1993; Kutsch and Schumacher, 1994) observed in EF patterns of HA or HS the presence of humic material that does not enter the gel. It could depend both on the presence of metal-HS complexes (De Nobili, 1988) or on the high MS of the material, unable to enter into the gel (Duxbury, 1989). Another possibility to explain this

4363

Fig. 2. Electrofocusing gel of whole HA and A, B, C + D fractions from Chernozem soil.

unclear behaviour is the interaction of HS with carriers ampholytes (Kutsch and Schumacher, 1994; Govi et al., 1994). To investigate the inability of this fraction to move into the gel, we performed the EF analysis on a PAA gel with more large pores (3.5% T), which theoretically allows to globular proteins with a nominal molecular weight approx. 106 Da to enter into the gel (Righetti, 1983), but the EF patterns of fractions A were identical to those obtained by the standard gel used (5% T). It means that the apparent MS of fraction A was >106 Da, or that there is an interaction of fraction A with the carriers ampholytes. Taking into account that the corresponding soil organic matter fractions A, B and C + D had an effective electrophoretic mobility (Cavani et al., 2003), the absence of the charge in fraction A is unlikely. In any case the high MS fractions A practically did not take part in the formation of the EF profile. The EF pattern of fraction B is characterised by a large and intense band not well focused in 4.5–8.0 pH region, and some bands with low intensity focused in 3.5–4.5 pH region. The fraction C + D shows several and intense bands focused in to the 3.5–5.7 pH region. In conclusion, the EF pattern of C + D fraction and whole HA were similar, because the fraction A does not contribute to the EF pattern, and fraction B focalise only in 4.5–8.0 pH region. These results show that, according to the theory of interpretation of EF migration/behaviour of HA, the fractions A and B with low charge-to-mass ratio (high pKa) do not migrate in the gel or they focalise in the high pH region (4.5–8.0). On the contrary, the fraction C + D with high charge-to-mass ratio (low pKa) focalises in the low pH region (3.5–5.7). The partially overlapping between the

4364

L. Cavani et al. / Bioresource Technology 99 (2008) 4360–4367

pH regions in witch focalise the fractions B and C + D is probably due to the heterogeneity of the fractions.

3.2.2. HS from compost EF densitograms of compost HA-like t0 and t130 and their fractions are shown in Figs. 3 and 4, respectively. By comparing the EF patterns of HA-like t0 and HA-like

Whole

absorbance at 633 nm

A0

B0

BCD0

C+D0

3.5

4.0

4.5

5.7

pH Fig. 3. Electrofocusing densitogram (k = 633 nm) of whole HA-like t0 and their fractions A, B, BCD and C + D at the beginning of the composting process.

Whole

absorbance at 633 nm

A130

B130

BCD130

C+D130

3.5

4.0

4.5

5.7

pH Fig. 4. Electrofocusing densitogram (k = 633 nm) of whole HA-like t130 and their fractions A, B, BCD and C + D after 130 days of the composting process.

t130 it was observed that the bulk HA-like t130 sample showed more bands in the 4.5–5.7 pH region, similarly with results formerly obtained (De Nobili et al., 1989; Garcia et al., 1992; Govi et al., 1993; Ciavatta et al., 1993a, 1993b). These findings probably reflect the evolution of compost’ organic matter and one of the explanation of the new bands with high apparent pI should be the formation of ex novo HS with low charge-to-mass ratio (Ciavatta et al., 1993a). Bands focused at pH > 4.5 were used as index to evaluate the compost quality (Canali et al., 1998). In our investigations high MS fractions A0 and A130 did not showed any peak corresponding to the bands focused at 3.5–8.0 pH region (Figs. 3 and 4), similar to the results obtained with the soil HA (Fig. 2). The EF patterns of fractions B0 and B130 showed some differences (Figs. 3 and 4). In the 3.5–4.5 pH region fractions B0 and B130 had several similar bands, while in the pH > 4.5 fraction B130 revealed some new bands not present in B0 sample. A similar results were also observed for the BCD fractions. It should be noted that the presence of broad and intense bands in 3.5–4.5 pH region of B0 and BCD0 sample probably reflected the effect of staining Coomassie Brilliant Blue R-250, which coloured the proteinous compounds contained in HA-like t0 at the beginning of composting (Senesi and Brunetti, 1996; Grigatti et al., 2004). In fact, before staining the visible brownish bands of B0 and BCD0 fractions were thin and better focused than after staining (Figs. 5 and 6). The absence of this effect in B130 and BCD130 samples is probably due to the degradation process of proteins that occurred during composting. The EF profiles of C + D0 and C + D130 fractions showed a deep transformation after composting. In fact, C + D0 pattern did not revealed any bands with the exclusion of a band on the anode (Fig. 3). On the contrary, the pattern of C + D130 fraction showed several bands principally focused in the 3.5–4.5 pH region; some bands are present in the 4.5–5.7 pH region, but with low intensity (Fig. 4). Moreover, it was possible to see an appreciable increased of the intensity of the band on the anode. This fraction showed intensive qualitative and quantitative changes in the EF pattern, and on the other hand the increase of relative quantity of this fraction in HA-like after composting have been observed as well (Table 2). It is possible to assume that during composting the high MS fractions was hydrolysed in more stable and less acidic low MS fragments. The EF pattern of compost HA-like t0 and t130 was formed by the sum of the fractions B and C + D. This profile revealed new bands in the 4.5–5.7 pH region after the composting process. The new bands are principally due to the high MS fraction B, but also the low MS fraction C + D showed some bands in this pH region. Probably, during composting the starting organic matter was degraded (hydrolysed) in more stable, low MS and less acidic compounds. The dissolved organic carbon (DOC), a parameter used to predict the compost maturity

L. Cavani et al. / Bioresource Technology 99 (2008) 4360–4367

4365

Fig. 5. Electrofocusing gel of whole HA-like t0, t130 and their fractions A, B, BCD, C + D.

Fig. 6. Electrofocusing gel coloured by Coomassie brilliant blue R-250 of whole HA-like t0, t130 and their fractions A, B, BCD, C + D.

(Zmora-Nahum et al., 2005) that decrease to 16.3– 6.2 g kg 1, seems to confirm this hypothesis.

4. Conclusions The evolution of compost humic acids-like (HA-like) and their fractions obtained by coupled SEC–PAGE at the beginning and after 130 days of the composting process have been characterised by electrofocusing (EF) and compared with HA of a Typic Chernozem soil. The results confirm that coupled SEC–PAGE is a suitable technique for preparative fractionation of HS. During composting each SEC–PAGE fraction was subjected to

modifications: the high MS fractions decrease, while the lower increase (i.e. C + D). The comparative analysis of EF profile of t0 vs. t130 unfractionated compost HA-like showed a significantly increase in the highest pH region (>4.5). The analysis of EF profile of SEC–PAGE fractions revealed that the high MS fractions practically did not take part in the EF pattern formation. Therefore, the variations observed in the EF patterns between the unfractionated HA-like were mainly due to the intermediate (B and BCD) and low MS fractions (C + D). It could be suggested that during composting the high MS fractions was partially degraded (or hydrolysed) and their higher apparent pI (>4.5) support the

4366

L. Cavani et al. / Bioresource Technology 99 (2008) 4360–4367

hypothesis of a partial rearrangement of these molecular fragments. Acknowledgement This research has been supported with funds provided by the INTAS (Ref. No. 05-1000008-8055). References Adani, F., Genevini, P.L., Gasperi, F., Zorzi, G., 1997. Organic matter evolution index (OMEI) as a measure of composting efficiency. Compost. Sci. Util. 5 (2), 53–62. Adani, F., Lozzi, P., Genevini, P., 2001. Determination of biological stability by oxygen uptake on municipal solid waste and derived products. Compost. Sci. Util. 9 (2), 163–178. Alianiello, F., 2003. Isoelectric focusing in soil science. J. Sept. Sci. 26, 387–391. Alianiello, F., Baroccio, F., 2004. Molecular weight fractions of soil and peat humic substances analysed by isoelectric focusing. Commun. Soil Sci. Plan. 35 (19–29), 53–62. Amir, S., Hafidi, M., Lemee, L., Merlina, G., Guiresse, M., Pinelli, E., Revel, J.-C., Bailly, J.-R., Ambles, A., 2006. Structural characterization of humic acids, extracted from sewage sludge during composting, by thermochemolysis-gas chromatography–mass spectrometry. Proc. Biochem. 41, 410–422. Barrena Go´mez, R., Va´quez Lima, F., Gordillo Bolasell, M.A., Gea, T., Sa´nchez Ferrer, A., 2005. Respirometric assay at fixed temperatures to monitor composting process. Bioresour. Technol. 96, 1153–1159. Brunetti, G., Plaza, C., Senesi, N., 2005. Olive pomace amendment in mediterranean conditions: Effect on soil and humic acid properties and wheat (Triticum turgidum L.) yield. J. Agric. Food Chem. 53, 6730– 6737. Cavani, L., Ciavatta, C., Trubetskaya, O.E., Afanas’eva, G.V., Reznikova, O.I., Trubetskoj, O.A., 2003. Capillary zone electrophoresis of soil humic acid fractions obtained by coupling SEC–PAGE. J. Chromatogr. A 938, 263–270. Canali, S., Trinchera, A., Pinzari, F., Benedetti, A., 1998. Study of compost maturity by means of humification parameters and isoelectrofocusing technique. In: Proceedings of the Transaction of the 16th World Congress of Soil Science. Montpellier, France, 20–26 August, Paper no. 778 (CD-ROM). Ceccanti, B., Bertolucci, M.T., Nannipieri, P., 1980. Characterisation of soil organic matter and derivate fraction by isoelectrofocusing. Rec. Develop. Chromat. Electroph. 10, 75–81. Chen, Y., Inbar, Y., 1993. Chemical and spectroscopical analysis of organic matter transformations during composting in relation to compost maturity. In: Hoitink, H.A.J., Keener, H.M. (Eds.), Science and Engineering of Composting: Design Environmental, Microbiological and Utilization Aspect. Renaissance Publications, Worthington, OH, USA, pp. 551–600. Ciavatta, C., Govi, M., 1993. In: Use of insoluble polyvinylpyrrolidone and isoelectric focusing in the study of humic substances in soils and organic wastes. J. Chromatogr. 643, 261–270. Ciavatta, C., Govi, M., Pasotti, L., Sequi, P., 1993a. Changes in organic matter during stabilisation of compost from municipal solid wastes. Bioresour. Technol. 43, 141–145. Ciavatta, C., Govi, M., Sequi, P., 1993b. Characterization of organic matter in compost produced with municipal solid wastes: an Italian approach. Compost. Sci. Util. 1, 75–81. De Nobili, M., 1988. Electrophoretic evidence of the integrity of humic substances separated by means of electrofocusing. J. Soil Sci. 39, 437– 445. De Nobili, M., Ciavatta, C., Sequi, P., 1989. Evaluation of organic matter stabilisation during composting by means of humification parameters and electrofocusing. In: Compost: Production and Use. Proceeding of

the International Symposium. San Michele all’Adige, Italy, pp. 328– 342, June 20–23. De Nobili, M., Bragato, G., Alcaniz, J.M., Puigbo, A., Comellas, L., 1990. Characterisation of electrophoretic fractions of humic substances with different electrofocusing behaviour. Soil Sci. 150, 763–770. De Nobili, M., Chen, Y., 1999. Size exclusion chromatography of humic substances: limits, perspectives and prospectives. Soil Sci. 164, 825– 833. Duxbury, J.M., 1989. Studies of the molecular size and charge of humic substances by electrophoresis. In: Hayes, M.B.H., Mac Carthy, P., Malcolm, R.L., Swift, R.S. (Eds.), Humic Substances II, In search of structure. Wiley, Chichester, pp. 593–620. Eckmeier, E., Gerlach, R., Gehrt, E., Schmidt, M., 2007. Pedogenesis of Chernozems in Central Europe. – A review. Geoderma 139, 288– 299. Fortun, C., Duato, M., 1986. Modificaciones que sufren las substancias humicas como consecuencia de la paja aplicada al suelo. Agrochimica 30, 358–367. Garcia, C., Herna`ndez, T., Costa, F., Ceccanti, B., Dell’Amico, C., 1992. Characterization of the organic fraction of an uncomposted and composted sewage sludge by isoelectric focusing and gel filtration. Biol. Fert. Soil 15, 112–118. Govi, M., Ciavatta, C., Gessa, C., 1993. Evolution of organic matter in sewage sludge: a study based on the use of humification parameter and analytical electrofocusing. Bioresour. Technol. 44, 175–180. Govi, M., Bonoretti, G., Ciavatta, C., Sequi, P., 1994. Characterization of soil organic matter using isoelectric focusing: a comparison of six commercial carrier ampholytes. Soil Sci. 157, 91–96. Grigatti, M., Ciavatta, C., Gessa, C., 2004. Evolution of organic matter from sewage sludge and garden trimming during composting. Bioresour. Technol. 91, 163–169. Grigatti M., Cavani L., Ciavatta C., 2006. A multivariate approach to the study of the composting process by means of analytical electrofocusing. Waste Management in press (doi:10.1016/j.wasman.2006.05.011). Kononova, M.M., 1966. Soil Organic Matter. Pergamon Press, Oxford. Kutsch, H., Schumacher, B., 1994. Isoelectric focusing of humic substances on ultrathin polyacrylamide gels: evidence of fingerprint performance. Biol. Fert. Soil 18, 163–167. Marmo, L., 2000. Towards a European strategy for biodegradable waste management. In: Ricicla 2000 (L. Morselli, a cura di). Maggioli Editore, Rimini, pp.21–30. Piccolo, A., 2001. The supramolecular structure of humic substances. Soil Sci. 166, 810–832. Requena, N., Azco´n, R., Baca, T.M., 1996. Chemical changes in humic substances from compost due to incubation with ligno-cellulolytic micro-organism and effect on lettuce growth. Appl. Microbiol. Biotech. 45, 857–863. Righetti, P.G., 1983. Isoelectric Focusing: Theory, Methodology and Applications. Elsevier, Amsterdam. Roletto, E., Chiono, R., Barberis, R., 1985. Investigation of humic matter from decomposting poplar bark. Agric.Wastes 12, 261–272. Senesi, N., Brunetti, G., 1996. Chemical and physico-chemical parameters for quality evaluation of humic substances produced during composting. In: The science of composting, London – Glasgow – Weinheim – New York – Tokio – Melbourne – Madras, pp. 195–212. Senesi, N., Plaza, C., Brunetti, G., Polo, A., 2007. A comparative survey of recent results on humic-like fractions in organic amendments effect on native soil humic substances. Soil Biol. Biochem. 39, 1244–1262. Trubetskaya, O.E., Trubetskoj, O.A., Ciavatta, C., 2001. Evaluation of the transformation of organic matter to humic substances in compost by coupling sec-page. Bioresour. Technol. 77, 51–56. Trubetskoj, O.A., Kudryavceva, L.Y., Shirshova, L.T., 1991. Characterization of soil humic matter by polyacrylamide gel electrophoresis in the presence of denaturing agents. Soil Biol. Biochem. 23, 1179–1181. Trubetskoj, O.A., Trubetskaya, O.E., Afanas’eva, G.V., Reznikova, O.I., Saiz-Jimenez, C., 1997. Polyacrylamide gel electrophoresis of soil

L. Cavani et al. / Bioresource Technology 99 (2008) 4360–4367 humic acid fractionated by size-exclusion chromatography and ultrafiltration. J. Chromatogr. A 767, 285–292. Trubetskoj, O.A., Trubetskaya, O.E., Afanas’eva, G.V., Reznikova, O.I., 1999. Weight and optical differences between soil humic acids fractions obtained by coupling SEC–PAGE. Geoderma 93 (3–4), 277–287.

4367

Tsiplenkov, B.P., 1983. Using of the isoelectric focusing for characterisation of soil humic matter. Vestnik Leningradskogo Universiteta 15, 106–108. Zmora-Nahum, S., Markovitch, O., Tarchitzky, J., Chen, Y., 2005. Dissolved organic carbon as a parameter of compost maturity. Soil Biol. Biochem. 37, 2109–2116.