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Identification of organic matter from peat, leonardite and lignite fertilisers using humification parameters and electrofocusing
Bioresource Technology 86 (2003) 45–52
Identification of organic matter from peat, leonardite and lignite fertilisers using humification parameters and electrofocusing L. Cavani *, C. Ciavatta, C. Gessa Department of Agroenvironmental Sciences and Technologies, University of Bologna, Viale Fanin n. 40, 40127 Bologna, Italy Received 6 December 2001; received in revised form 18 April 2002; accepted 24 April 2002
Abstract The organic matter extracted from peats (P), leonardites (Le) and lignites (Li) was characterised by humification parameters and electrofocusing (EF). The degree of humification and the humification index might be used to distinguish P from Le and Li, but not Le from Li because they showed overlapped values, while the humification rate could be used only for the identification of Le and EF profiles of P, Le and Li fertilisers revealed different band patterns: P samples did not show bands in the region with isoelectric point, pI > 4:4; Le samples showed very intense bands in the region with pI > 4:4; Li samples showed a very different band pattern with poorly resolved bands in the region with pI > 3:8. P, Le and Li samples can be distinguished by combining humification parameters and EF. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Degree of humification; Humification rate; Humification index; Electrofocusing; Peat; Leonardite; Lignite; Fertilisers
1. Introduction The chemical characterisation of the organic matter extracted from peats (P), leonardites (Le) and lignites (Li) is difficult because of the complexity of humic substances (HS) and the great heterogeneity of the materials. Organic matter from these materials containing a high amount of HS has been characterised using spectroscopic techniques, such as laser fluorescence (Morita and Measures, 1983), 13 C-NMR (US Geological Survey, 1989), 1 H-NMR (US Geological Survey, 1989), FT-IR (Francioso et al., 1996, 1998), surface enhanced Raman spectroscopy (S anchez-Cortez et al., 1998). However, it usually happens for the HS from different origins, that these techniques show some difficulties in characterising the organic matter. Ciavatta et al. (1989) proposed the use of two humification parameters, the degree of humification (DH) and the humification rate (HR), to evaluate the humification level in organic materials. Since then, DH and HR have been applied to several organic materials, such as soils (Govi et al., 1992), slurries (Govi et al., 1989), compost (De Nobili et al., 1989; *
Corresponding author. Tel.: +39-051-2096209; fax: +39-0512096203. E-mail address: [email protected] (L. Cavani).
Ciavatta et al., 1992; Govi et al., 1993b), and sludges (Govi et al., 1993a). Humification parameters have also been applied to characterise organic and organic-mineral fertilisers (Ciavatta et al., 1989, 1996; Govi et al., 1989; Alianiello et al., 1999) and appeared to be useful for the differentiation of P from Le samples, but not for their humic extracts (HE) (Ciavatta et al., 1996). De Nobili et al. (1985) proposed the use of gel electrofocusing (EF), as a qualitative parameter to characterise HS of different origins and to evaluate the humification level of an organic material from a qualitative point of view. Govi et al. (1992) showed that the EF technique was useful for evaluation of differences in soil organic matter not detectable by quantitative parameters such as DH and HR. In a previous work Ciavatta et al. (1996) used EF to characterise and distinguish P from Le samples and HE prepared from P or Le. The authors demonstrated that in the region with pH gradient over pH 5 of the EF profiles of Le and HE from Le, there was a poorly resolved group of bands with a very high intensity. This group of bands was not present in the EF profiles of P and HE from P samples or, if present, it was much less intense than that in the EF profiles of Le and of HE from Le (Ciavatta et al., 1996). Despite the interesting results obtained, Ciavatta et al. (1996) were not able to make a complete EF characterisation of the organic
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materials studied due to the unresolved group of bands focused over pH 5. This problem could probably be due to two factors: (i) the reticulation degree of the polyacrylamide (PAA) gel and (ii) the prerun time. It has been shown (Ceccanti et al., 1980; Ciavatta et al., 1996) that in the EF fractionates the apparent isoelectric point (pI) of the humic compounds increases with the increasing of their nominal molecular weight (NMW). The reticulation of the PAA used by Ciavatta et al. (1996) was given by a 29:1 acrylamide:bis-acrylamide (3.33%C, 5%T). These conditions permit free migration of the organic compounds with a NMW lower than about 200 kDa (Righetti, 1983). Tonelli et al. (1997), showed that after fractionation using a tangential ultrafiltration system, the NMW of a HS fraction extracted from P sample was higher than 300 kDa, so it is reasonable to suppose that the NMW of a HS fraction from Le or Li also contains organic compounds with NMW much higher than 300 kDa. These compounds with a high NMW could not move freely during an EF run conducted in the conditions used by Ciavatta et al. (1996). In a previous work Duxbury (1989) showed that the resolution of HS in a gel EF fraction was a function of the prerun time. A too short prerun did not permit the complete prefocusing of the carrier ampholytes and the preformation of the pH gradient. HS with a high apparent pI can migrate towards the anode, without focus, at the corresponding pH value. The aim of this work was (i) to apply the humification parameters to several samples of P, Le, and Li; (ii) to propose an EF method for a better characterisation of the organic matrices of P, Le and Li samples and (iii) to determine if this method could be applied to the identification of the matrix of P, Le and Li.
atmosphere. After extraction the samples were centrifuged at 5000 g for 15 min and the supernatants were filtered through a 0.8 lm filter (Millipore, USA). This filtered solution constituted the total extract (TE) and was fractionated into humified (humic, HA þ fulvic acids, FA) and non-humified (NH) fractions (Ciavatta et al., 1990). Briefly, 25 ml of TE was putt into a 50 ml centrifuge tube and acidified to pH < 2 by adding a small volume (0.3–0.5 ml) of 9 M H2 SO4 , then centrifuged at 5000g for 20 min. The precipitated fraction (HA) was collected and stored, and the supernatant solution fed onto a small column packed with about 5 cm3 of insoluble polyvinylpyrrolidone, previously equilibrated in 0.005 M H2 SO4 . The eluate (NH) was collect in a 50 ml volumetric flask, then diluted to volume with 0.005 M H2 SO4 and stored. The retained fraction (FA) was eluted with 0.5 M NaOH solution and collected in the centrifuge tube containing the HA precipitate, which redissolved. Total organic carbon (TOC), total extracted carbon (TEC) and humified carbon (HA þ FA) were determined according to dichromate acid oxidation methods (Ciavatta et al., 1989, 1991). The DH (Ciavatta et al., 1988), the HR (Ciavatta et al., 1988) and the humification index (HI) (Sequi et al., 1986) were calculated as follow: DH% ¼ 100
ðHA þ FAÞ TEC
HR% ¼ 100
ðHA þ FAÞ TOC
HI ¼
NH ðHA þ FAÞ
2.3. Electrofocusing 2. Methods 2.1. Fertiliser samples The 29 samples of P (8 standard references þ 21 commercial products), 11 samples of Le (4 standard references þ 7 commercial products), 20 samples of HE from Le (4 standard references þ 16 commercial products) and 5 samples of Li (standard references only) used in this study were taken from the collection of the Department of Agroenvironmental Sciences and Technologies of the University of Bologna. Chemical characteristics of the samples are reported in Tables 1–4. 2.2. Humification parameters Organic carbon was extracted by shaking 2 g of sample in a 250 ml centrifuge tube with 100 ml of 0.1 M NaOH plus 0.1 M Na4 P2 O7 10 H2 O for 48 h at 65 C and 120 rpm in a thermostatic water bath under N2
About 25 ml of TE were buffered at pH 7 with 1 M H2 SO4 , dialysed in a 1000 daltons cut-off dialysis tube (Cellu Sep H1, Membrane Filtration Products Inc., USA) against deionised water, at 2 C, then freeze dried, redissolved in double distilled water (5 mg ml1 of lyophilised sample) and then frozen. The EF separations were carried out in a 5%T (the total monomer concentration (%T) ¼ (g acrylamide þ g bis-acrylamide)/total volume 100) and 3.33%C (the crosslinking monomer concentration (%C) ¼ g bisacrylamide/g acrylamide þ g bis-acrylamide)) (Ciavatta et al., 1996) or 2.6%C PAA slab gels (Bio-Rad Laboratories, USA), containing 2% of carrier ampholytes (CA’s). The CA’s used were composed of a mixture of 60% Ampholine (Pharmacia Biotech, Sweden) pH gradient from 3.5 to 5.0, 20% Ampholine with a pH gradient from 4.0 to 6.0 and 20% Ampholine with a pH gradient from 6.0 to 8.0 (Ciavatta et al., 1996). The slabs (260 110 0:5 mm) were prerun for 1, 2, 3 or 4 h in an electrophoretic cell (Multiphore II, LKB, Sweden),
L. Cavani et al. / Bioresource Technology 86 (2003) 45–52
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Table 1 Characteristics of peat samples Samples
Origin
pHa
Standard (P s) 1 2 3 4 5 6 7 8
Ireland Italy Italy Italy Italy Germany Germany Russia
4.8 7.3 7.1 5.7 6.5 4.8 3.6 2.5
Commercial (P c) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Czech Rep. Ireland Ireland Ireland Ireland Ireland Italy Italy Italy Italy Italy Italy Norway Germany Germany Germany Russia Russia Russia Russia Ukraine
6.2 5.3 3.5 3.5 3.5 3.6 7.3 6.4 6.8 6.8 6.8 6.1 4.6 3.0 5.4 5.0 2.5 3.0 4.1 4.5 4.6
Moistureb (%)
Ashc (%)
TOC (%)
TEC (%)
HA þ FA (%)
4.0 10.6 4.5 8.5 8.9 9.1
8.8 74.3 65.9 15.3 57.4 2.7 0.8 6.2
52.3 12.4 20.7 41.9 21.5 49.3 49.2 47.2
26.3 7.6 12.8 26.1 16.1 21.0 21.5 31.0
23.6 5.9 10.4 22.1 13.6 18.2 17.3 25.8
8.9 10.2 9.6 8.6 10.2 10.3 5.1 7.1 9.8 6.4 7.1 17.3 8.5 12.0 6.9 9.6 8.2 9.6 8.6 11.6 9.3
12.2 1.9 8.3 2.0 1.3 2.3 64.7 60.0 42.4 44.1 58.3 53.3 6.3 1.3 14.6 8.2 7.2 8.4 7.6 19.2 21.5
45.1 50.4 47.4 50.6 49.9 47.6 20.9 20.5 26.5 28.1 21.3 20.7 51.7 42.3 43.7 45.7 48.1 41.8 51.2 40.6 39.5
25.3 39.8 24.6 31.7 32.1 17.9 11.4 14.9 18.2 19.6 13.6 14.6 34.2 15.0 21.7 21.2 34.9 21.6 26.4 30.2 23.7
22.1 27.8 21.6 28.4 29.1 14.8 9.6 12.3 16.5 18.0 11.0 12.2 29.6 11.0 19.0 19.1 31.8 16.8 25.7 26.5 21.0
7.1 3.2
a
Sample water ratio 1:10. Wet matter. c Dry matter. b
Table 2 Characteristics of leonardite samples Samples
Origin
pHa
Moistureb (%)
Ashc (%)
TOC (%)
TEC (%)
HA þ FA (%)
Standard (Le s) 1 2 3 4
USA USA USA USA
3.8 9.6 3.5 4.1
13.5 11.6 14.5 11.8
20.2 40.0 21.3 21.8
43.8 39.9 41.4 41.8
36.5 39.8 29.1 32.7
35.8 35.6 28.3 28.9
Commercial (Le c) 1 2 3 4 5 6 7
Spain Spain USA USA Unknown Unknown Unknown
2.9 5.5 3.7 3.8 4.6 3.6 5.1
7.7 11.2 12.8 18.4 11.6 5.7 9.8
60.8 42.7 16.0 21.0 20.4 72.5 29.7
21.2 31.2 45.4 39.1 48.2 15.7 40.4
14.6 24.4 34.9 32.0 43.6 10.9 25.1
12.2 22.6 33.5 30.6 42.0 9.9 22.6
a
Sample water ratio 1:10. Wet matter. c Dry matter. b
cooled at 2 C and supplied by a 2197 Power Supply (LKB, Sweden). The distance between the two electrodes was about 90 mm. The prerun and run conditions
were: max voltage 1200 V, power 0.9 W cm1 and current 0.6 mA cm1 . After loading an aliquot of sample (25–50 ll) the run was conducted for 2 h. After each run
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L. Cavani et al. / Bioresource Technology 86 (2003) 45–52
Table 3 Characteristics of humic extract of leonardite samples Samples
Origin
pHa
Moistureb (%)
Ashc (%)
TOC (%)
TEC (%)
HA þ FA (%)
Standard (HE s) 1 2 3 4
Spain USA USA USA
10.2 11.9 9.5 8.7
75.0 77.0 86.3 15.1
48.4 48.9 51.5 42.1
9.8 10.0 5.4 37.6
8.3 8.6 4.3 30.9
8.2 7.3 4.0 30.5
4.1 8.0 5.9 7.3 8.7 9.2 9.9 9.4 9.3 10.5 10.5 11.3 11.4 9.7 9.3 9.3
13.0 14.1 11.0 12.0 19.6 92.3 12.1 86.8 85.3 11.6 10.2 64.3 64.2 14.3 81.5 75.3
34.0 42.8 35.4 37.7 60.8 41.8 41.1 39.4 45.7 57.9 42.5 52.7 53.5 46.6 49.1 43.4
28.8 26.8 30.7 29.2 27.0 3.5 30.6 3.6 6.2 22.2 31.9 12.7 12.6 39.8 8.0 11.0
28.9 24.5 30.1 27.8 25.9 2.9 24.3 3.4 5.0 19.3 17.3 10.0 11.3 29.7 6.4 8.6
26.5 22.9 28.1 25.7 24.3 2.8 23.2 2.9 4.4 17.3 14.7 9.5 10.9 27.6 6.2 7.5
Commercial (HE c) 1 USA 2 USA 3 USA 4 USA 5 USA 6 USA 7 USA 8 USA 9 USA 10 USA 11 USA 12 USA 13 USA 14 Unknown 15 Unknown 16 Unknown a
Sample water ratio 1:10. Wet matter. c Dry matter. b
Table 4 Characteristics of lignite samples Samples
Origin
pHa
Moistureb (%)
Ashc (%)
TOC (%)
TEC (%)
HA þ FA (%)
Standard (Li) 1 2 3 4 5
Hungary Hungary Hungary Hungary USA
5.8 2.8 5.0 6.5 6.5
21.8 9.6 19.2 16.0 13.5
14.9 3.9 27.1 18.0 8.7
47.0 62.1 40.8 49.6 53.6
27.0 35.9 26.6 31.4 17.6
26.9 34.5 25.5 30.9 8.3
a
Sample water ratio 1:10. Wet matter. c Dry matter. b
the slabs were stained for two, under a gentle continuous stirring, in a solution containing 15% CH3 COOH (glacial, 100%), 15% CH3 CH2 OH (95%), 0.025% Blue Brilliant R-250 Coomassie (Merck, Germany) and 1% CuSO4 (99.5%). Then the slabs were treated overnight, under a gentle continuous stirring, with a solution containing: 15% CH3 COOH, 15% CH3 CH2 OH, 0.0025% Blue Brilliant R-250 Coomassie and 1% CuSO4 and finally with a solution prepared with 10% CH3 COOH and 10% CH3 CH2 OH to a complete destaining of the areas with no bands. After destaining the focused bands were scanned at 633 nm with an Ultroscan XL Enhanced Laser Densitometer (Pharmacia Biotech, Sweden). The bands of the EF densitograms were integrated and evaluated using the 2.1 version of the Gelscan software (Pharmacia Biotech, Sweden).
3. Results and discussion 3.1. Humification parameters The amounts of TOC, TEC and humificated carbon (HA þ FA) are reported in Tables 1–4. As with the results reported by Ciavatta et al. (1996) the TOC, TEC and HA þ FA differed both in standard and in commercial samples (Tables 1–4) and therefore could not be used to distinguish P from Le and Li samples. In the Figs. 1–3 are reported the distributions of calculated humification parameters. In spite of the heterogeneity of both matrices, due to the different origins and chemical properties (Tables 1–4), the humification parameters were able to characterise the different fertilisers. In fact, the differences between standard and
L. Cavani et al. / Bioresource Technology 86 (2003) 45–52
Fig. 1. Box plot of DH of fertiliser samples (P t: peats total, P s: peats standard, P c: peats commercial, Le t: leonardites total, Le s: leonardites standard, Le c: leonardites commercial, HE t: humic extracts of leonardite total, HE s: humic extracts of leonardite standard, HE c: humic extracts of leonardite commercial, Li: lignites).
49
Fig. 3. Box plot of HI of fertiliser samples (P t: peats total, P s: peats standard, P c: peats commercial, Le t: leonardites total, Le s: leonardites standard, Le c: leonardites commercial, HE t: humic extracts of leonardite total, HE s: humic extracts of leonardite standard, HE c: humic extracts of leonardite commercial, Li: lignites).
Nevertheless, the DH and the HI may only be used to distinguish P (DH < 90%, HI > 0:15) from Le and Li (Figs. 1 and 3), and may not be used to distinguish Le (DH > 90%; HI < 0:15) from Li (DH > 90%, HI < 0:12), because they show overlapping values. Because the HR found in P samples (Fig. 2) was lower than 65% while it was higher than 65% in Le samples it has been suggested (Ciavatta et al., 1996) that HR be used to distinguish P from Le samples. However, the mean HR of the Li samples (Fig. 2) differs significantly from those of the P samples for p ¼ 0:004 (Student t-test), but several samples of P showed an HR value similar to that of the Li samples (Fig. 2), and, finally, the number of Li samples was relatively limited (Table 4). On the basis of these results it is reasonable to suggest that HR may not be used for the identification of P from Le and Li, HR may only be used for identification of Le (HR > 65%). 3.2. Electrofocusing Fig. 2. Box plot of HR of fertiliser samples (P t: peats total, P s: peats standard, P c: peats commercial, Le t: leonardites total, Le s: leonardites standard, Le c: leonardites commercial, HE t: humic extracts of leonardite total, HE s: humic extracts of leonardite standard, HE c: humic extracts of leonardite commercial, Li: lignites).
commercial fertilisers were not significant for each type of fertiliser (Figs. 1–3), but the commercial products showed the most amount of data outside the 10th and 90th percentile.
In order to simplify the interpretation of the results obtained, the EF profiles have been subdivided into three main regions (Figs. 4–6): 1. region A, from pH 3.5 to about pH 3.8, characterised by a very intense band at a low apparent pI and by some other, not intense, bands; 2. region B, from pH 3.8 to pH 4.4, characterised by a group of four very well-resolved bands; 3. region C, with a group of bands focused over pH 4.4.
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L. Cavani et al. / Bioresource Technology 86 (2003) 45–52
Fig. 4. Typical EF scan profiles at 633 nm of peat samples (P s: peats standard, P c: peats commercial), each sample is identified by a number detailed in Table 1.
Fig. 5. Typical EF scan profiles at 633 nm of leonardite and HE of leonardite samples (Le s: leonardite standard, Le c: leonardite commercial, HE: humic extract of leonardite), each sample is identified by a number detailed in Tables 2 and 3.
The EF profiles of P samples (Fig. 4) were characterised by two main groups of bands in the regions A and B, and sometimes by a third group of faint bands in the region near to the cathode (region C). Region B is the most important one, where it was possible to identify four bands (Fig. 4) with different intensities: bands 1–3 had a similar relative area, while band 4 had a bigger relative area. In some samples the area of band 4 was
Fig. 6. Typical EF scan profiles at 633 nm of lignite samples (Li: lignite), each sample is identified by a number detailed in Table 4.
similar to the areas of bands 1–3 (Fig. 4, low humified), while in some samples the area of band 4 was bigger than that of the bands 1–3 (Fig. 4, normally humified). The area of band 4 is very important because it corresponds to the humification level of P. In fact, several authors (De Nobili et al., 1990; Ciavatta and Govi, 1993; Kutsch and Schumacher, 1994) showed that the most complex and stable humic compounds are characterised by a pI higher than humic compounds with a lower humification level. Some of the samples that presented a big area in band 4 of the EF profile were also characterised by the presence of some bands in region C suggesting a higher humification level (Fig. 4, high humified). This result is apparently not in agreement with those reported by Ciavatta et al. (1996) where several P organic extracts showed a group of bands in this region. Ciavatta et al. (1996), however, used a 3.3%C PAA gel that was less porous than the 2.6%C PAA gel used in this work. It is reasonable to suppose that the organic compounds with higher molecular size were not able to run in the gel used by Ciavatta et al. (1996) and that they could not reach their focusing point. The EF profile of standard Le, commercial Le and HE of Le is shown in Fig. 5. The EF profiles of Le and HE are similar to P profiles in regions A and B, but in region C these profiles are characterised by a very intense group of bands (Fig. 5). This finding is in agreement with the hypothesis that the Le were composed of the most humified substances with an high apparent pI (pH > 4:4) (De Nobili et al., 1990). Band 4 of region B is more intense than bands 2 and 3 (Fig. 5), and the area of region C is 3–4 times bigger than the area of region B (Table 5). The EF profiles of HE samples are similar to
L. Cavani et al. / Bioresource Technology 86 (2003) 45–52 Table 5 Relative area (%) of bands from regions A, B and C calculated on the EF profiles at 633 nm Samples
n
Region A pH 3.5–3.8
Region B pH 3.8–4.4
Region C pH 4.4–6.0
P total P standard P commercial Le total Le standard Le commercial HE Le total HE Le standard HE Le commercial Li standard
29 8 21 11 4 7 20 4 16 5
51.7 9.5a 53.8 9.6 50.9 9.5 12.7 5.1 12.8 4.6 12.6 5.8 29.0 9.3 24.1 9.2 30.2 9.2 7.6 0.9
45.3 7.1 42.5 5.5 46.4 7.4 18.4 3.5 17.3 2.1 19.0 4.1 25.9 6.6 20.8 6.9 27.2 6.1 11.3 4.1
3.0 8.4 3.7 7.9 2.7 8.8 68.9 6.7 69.9 4.4 68.4 8.0 45.1 13.0 55.1 16.0 42.6 11.5 81.0 4.6
a
51
presence in region C of a very intense group of bands (relative area > 60%); • Li: HR ¼ 55–65%; absence or limited presence of bands 3–4 in region B; presence in region C of a poorly resolved group of bands (relative area > 75%). In conclusion, it is can by said that humification parameters (particularly HR) and EF are very useful for characterisation and identification of P, Le and Li in fertiliser samples.
Acknowledgements
Mean standard deviation.
EF profiles of Le samples (Fig. 5), the only difference is the relative area of region C; nearly 1.5–2 times bigger than the area of region B (Table 5). This reduction of materials with high apparent pI was probably due to the extraction processes. The EF profiles of Li samples (Fig. 6) was very different from those of P and Le, these profiles were characterised by a typical group of peaks with very low intensity in region B (only bands 3 and 4 were eventually present), and a poorly resolved group of bands in the region C: the integrated area of region C is 7–8 time greater than the area of region B (Table 5).
4. Conclusions Only partial characterisation of P, Le and Li is possible with the humification parameters: with HR is possible to distinguish Le from P and Li (Fig. 2), but not P from Li; with DH and HI it is possible to distinguish P from Le and Li (Figs. 1 and 3), but not Le from Li. The EF, instead, allows a precise characterisation of different fertilisers, and can distinguish between the different degrees of humification of P samples. On the base of the results shown here it is possible to propose the following classification: • P with a low humification level: HR < 40%, and/or absence of EF band 4 in region B; absence of bands in region C (relative area < 5%); • normally humified P: HR > 40%; presence of EF bands 1–4 in region B with similar intensity; absence of bands in region C (relative area < 5%); • very well humified P: HR > 50%; presence of EF bands 1–4. Bands 3 and 4 more intense than bands 1 and 2; bands in region C eventually present (relative area > 5%); • Le: HR > 65%; presence of EF bands 1–4 in region B; bands 1 and 4 more intense than bands 2 and 3;
This research was supported with funds provided by ‘‘Ispettorato Centrale Repressioni Frodi’’ of the Italian Ministry of the Agricultural and Forestry Politics (MiPAF). The authors thank Dr. Marco Govi for his help and valuable suggestions to develop this research.
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Giovannozzi-Sermanni, G., Sequi, P. (Eds.), Current Perspective in Environmental Biochemistry. Proceeding of a VIIth International Symposium of Environmental Biogeochemistry. CNR-IPRA, Rome, pp. 85–94. De Nobili, M., Ciavatta, C., Sequi, P., 1989. Evaluation of organic matter stabilization during composting by means of humification parameters and electrofocusing. In: Compost: Production and Use. Proceeding of the International Symposium, San Michele all’Adige, Italy, 20–23 June 1989. pp. 328–342. Duxbury, J.M., 1989. Studies of the molecular size and charge of humic substances by electrophoresis. In: Hayes, M.H.B., MacCarthy, P., Malcom, R.L., Swift, R.S. (Eds.), Humic Substances II, In Search of Structure. Wiley, Chicester, pp. 593–620. Francioso, O., S anchez-Cortez, S., Tugnoli, V., Ciavatta, C., Gessa, C., 1998. Characterisation of peat fulvic acid fractions by means of FT-IR, SERS and 1 H, 13 C NMR spectroscopy. Appl. Spectrosc. 52, 270–277. Francioso, O., S anchez-Cortez, S., Tugnoli, V., Ciavatta, C., Sitti, L., Gessa, C., 1996. Infrared, Raman, and nuclear magnetic resonance (1 H, 13 C and 31 P) spectroscopy in the study of fractions of peat humic acids. Appl. Spectrosc. 50, 1165–1174. Govi, M., Ciavatta, C., Gessa, C., 1993a. Evolution of organic matter in sewage sludge: a study based on the use of humification parameter and analytical electrofocusing. Bioresour. Tecnol. 44, 175–180. Govi, M., Ciavatta, C., Pasotti, L., Sequi, P., 1993b. Changes in organic matter during stabilization of compost from municipal solid wastes. Bioresour. Tecnol. 43, 141–145. Govi, M., Ciavatta, C., Vittori Antisari, L., Sequi, P., 1989. Characterization of organic materials by means of electrofocusing. In: Hallard, B., Boren, H., Grinvall, A. (Eds.), Humic Substances in
Aquatic and Terrestrial Environment. Proceeding of International Symposium, Link€ oping, Sweden, 21–23 August 1989. SpringerVerlag, Berlin. pp. 143–149. Govi, M., Francioso, O., Ciavatta, C., Sequi, P., 1992. Influence of long-term residue and fertilizer applications on soil humic substances: a study by electrofocusing. Soil Sci. 154, 8–13. Kutsch, H., Schumacher, B., 1994. Isoelectric focusing of humic substances on ultrathin polyacrilamide gels: evidence of fingerprint performance. Biol. Fertil. Soils 18, 163–167. Morita, H., Measures, R.M., 1983. Some observations on the laser fluorescence spectroscopy of peats. In: Fuchsman, C.H., Spigarelli, S.A. (Eds.), International Symposium on Peat Utilisation, Bemidji, MN, USA, 10–13 October 1983. Bemidji State University, Bemidji. pp. 391–403. Righetti, P.G., 1983. Isoelectric Focusing: Theory, Methodology and Applications. Elsevier, Amsterdam. Sanchez-Cortez, S., Francioso, O., Ciavatta, C., Garcıa-Ramos, J.V., Gessa, C., 1998. pH-dependent adsorption of fractionated peat humic substances on different silver colloids studied by surfaceenhanced Raman spectroscopy. J. Colloid Interf. Sci. 198, 308–318. Sequi, P., De Nobili, M., Leita, L., Cercignani, G., 1986. A new index of humification. Agrochimica 30, 175–179. Tonelli, D., Seeber, R., Ciavatta, C., Gessa, C., 1997. Extraction of humic acids from a natural matrix by alkaline pyrophosphate. Evaluation of the molecular weight of fractions obtained by ultrafiltration. Fresenius J. Anal. Chem. 359, 555–560. US Geological Survey, 1989. Characterisation of the international humic substances society standard and reference fulvic and humic acids by solution state carbon-13 (13 C) and hydrogen-1 (1 H) nuclear magnetic resonance spectrometry. Water Resource Investigation Report 89-4196.
Identification of organic matter from peat, leonardite and lignite fertilisers using humification parameters and electrofocusing L. Cavani *, C. Ciavatta, C. Gessa Department of Agroenvironmental Sciences and Technologies, University of Bologna, Viale Fanin n. 40, 40127 Bologna, Italy Received 6 December 2001; received in revised form 18 April 2002; accepted 24 April 2002
Abstract The organic matter extracted from peats (P), leonardites (Le) and lignites (Li) was characterised by humification parameters and electrofocusing (EF). The degree of humification and the humification index might be used to distinguish P from Le and Li, but not Le from Li because they showed overlapped values, while the humification rate could be used only for the identification of Le and EF profiles of P, Le and Li fertilisers revealed different band patterns: P samples did not show bands in the region with isoelectric point, pI > 4:4; Le samples showed very intense bands in the region with pI > 4:4; Li samples showed a very different band pattern with poorly resolved bands in the region with pI > 3:8. P, Le and Li samples can be distinguished by combining humification parameters and EF. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Degree of humification; Humification rate; Humification index; Electrofocusing; Peat; Leonardite; Lignite; Fertilisers
1. Introduction The chemical characterisation of the organic matter extracted from peats (P), leonardites (Le) and lignites (Li) is difficult because of the complexity of humic substances (HS) and the great heterogeneity of the materials. Organic matter from these materials containing a high amount of HS has been characterised using spectroscopic techniques, such as laser fluorescence (Morita and Measures, 1983), 13 C-NMR (US Geological Survey, 1989), 1 H-NMR (US Geological Survey, 1989), FT-IR (Francioso et al., 1996, 1998), surface enhanced Raman spectroscopy (S anchez-Cortez et al., 1998). However, it usually happens for the HS from different origins, that these techniques show some difficulties in characterising the organic matter. Ciavatta et al. (1989) proposed the use of two humification parameters, the degree of humification (DH) and the humification rate (HR), to evaluate the humification level in organic materials. Since then, DH and HR have been applied to several organic materials, such as soils (Govi et al., 1992), slurries (Govi et al., 1989), compost (De Nobili et al., 1989; *
Corresponding author. Tel.: +39-051-2096209; fax: +39-0512096203. E-mail address: [email protected] (L. Cavani).
Ciavatta et al., 1992; Govi et al., 1993b), and sludges (Govi et al., 1993a). Humification parameters have also been applied to characterise organic and organic-mineral fertilisers (Ciavatta et al., 1989, 1996; Govi et al., 1989; Alianiello et al., 1999) and appeared to be useful for the differentiation of P from Le samples, but not for their humic extracts (HE) (Ciavatta et al., 1996). De Nobili et al. (1985) proposed the use of gel electrofocusing (EF), as a qualitative parameter to characterise HS of different origins and to evaluate the humification level of an organic material from a qualitative point of view. Govi et al. (1992) showed that the EF technique was useful for evaluation of differences in soil organic matter not detectable by quantitative parameters such as DH and HR. In a previous work Ciavatta et al. (1996) used EF to characterise and distinguish P from Le samples and HE prepared from P or Le. The authors demonstrated that in the region with pH gradient over pH 5 of the EF profiles of Le and HE from Le, there was a poorly resolved group of bands with a very high intensity. This group of bands was not present in the EF profiles of P and HE from P samples or, if present, it was much less intense than that in the EF profiles of Le and of HE from Le (Ciavatta et al., 1996). Despite the interesting results obtained, Ciavatta et al. (1996) were not able to make a complete EF characterisation of the organic
0960-8524/03/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 1 0 7 - 4
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L. Cavani et al. / Bioresource Technology 86 (2003) 45–52
materials studied due to the unresolved group of bands focused over pH 5. This problem could probably be due to two factors: (i) the reticulation degree of the polyacrylamide (PAA) gel and (ii) the prerun time. It has been shown (Ceccanti et al., 1980; Ciavatta et al., 1996) that in the EF fractionates the apparent isoelectric point (pI) of the humic compounds increases with the increasing of their nominal molecular weight (NMW). The reticulation of the PAA used by Ciavatta et al. (1996) was given by a 29:1 acrylamide:bis-acrylamide (3.33%C, 5%T). These conditions permit free migration of the organic compounds with a NMW lower than about 200 kDa (Righetti, 1983). Tonelli et al. (1997), showed that after fractionation using a tangential ultrafiltration system, the NMW of a HS fraction extracted from P sample was higher than 300 kDa, so it is reasonable to suppose that the NMW of a HS fraction from Le or Li also contains organic compounds with NMW much higher than 300 kDa. These compounds with a high NMW could not move freely during an EF run conducted in the conditions used by Ciavatta et al. (1996). In a previous work Duxbury (1989) showed that the resolution of HS in a gel EF fraction was a function of the prerun time. A too short prerun did not permit the complete prefocusing of the carrier ampholytes and the preformation of the pH gradient. HS with a high apparent pI can migrate towards the anode, without focus, at the corresponding pH value. The aim of this work was (i) to apply the humification parameters to several samples of P, Le, and Li; (ii) to propose an EF method for a better characterisation of the organic matrices of P, Le and Li samples and (iii) to determine if this method could be applied to the identification of the matrix of P, Le and Li.
atmosphere. After extraction the samples were centrifuged at 5000 g for 15 min and the supernatants were filtered through a 0.8 lm filter (Millipore, USA). This filtered solution constituted the total extract (TE) and was fractionated into humified (humic, HA þ fulvic acids, FA) and non-humified (NH) fractions (Ciavatta et al., 1990). Briefly, 25 ml of TE was putt into a 50 ml centrifuge tube and acidified to pH < 2 by adding a small volume (0.3–0.5 ml) of 9 M H2 SO4 , then centrifuged at 5000g for 20 min. The precipitated fraction (HA) was collected and stored, and the supernatant solution fed onto a small column packed with about 5 cm3 of insoluble polyvinylpyrrolidone, previously equilibrated in 0.005 M H2 SO4 . The eluate (NH) was collect in a 50 ml volumetric flask, then diluted to volume with 0.005 M H2 SO4 and stored. The retained fraction (FA) was eluted with 0.5 M NaOH solution and collected in the centrifuge tube containing the HA precipitate, which redissolved. Total organic carbon (TOC), total extracted carbon (TEC) and humified carbon (HA þ FA) were determined according to dichromate acid oxidation methods (Ciavatta et al., 1989, 1991). The DH (Ciavatta et al., 1988), the HR (Ciavatta et al., 1988) and the humification index (HI) (Sequi et al., 1986) were calculated as follow: DH% ¼ 100
ðHA þ FAÞ TEC
HR% ¼ 100
ðHA þ FAÞ TOC
HI ¼
NH ðHA þ FAÞ
2.3. Electrofocusing 2. Methods 2.1. Fertiliser samples The 29 samples of P (8 standard references þ 21 commercial products), 11 samples of Le (4 standard references þ 7 commercial products), 20 samples of HE from Le (4 standard references þ 16 commercial products) and 5 samples of Li (standard references only) used in this study were taken from the collection of the Department of Agroenvironmental Sciences and Technologies of the University of Bologna. Chemical characteristics of the samples are reported in Tables 1–4. 2.2. Humification parameters Organic carbon was extracted by shaking 2 g of sample in a 250 ml centrifuge tube with 100 ml of 0.1 M NaOH plus 0.1 M Na4 P2 O7 10 H2 O for 48 h at 65 C and 120 rpm in a thermostatic water bath under N2
About 25 ml of TE were buffered at pH 7 with 1 M H2 SO4 , dialysed in a 1000 daltons cut-off dialysis tube (Cellu Sep H1, Membrane Filtration Products Inc., USA) against deionised water, at 2 C, then freeze dried, redissolved in double distilled water (5 mg ml1 of lyophilised sample) and then frozen. The EF separations were carried out in a 5%T (the total monomer concentration (%T) ¼ (g acrylamide þ g bis-acrylamide)/total volume 100) and 3.33%C (the crosslinking monomer concentration (%C) ¼ g bisacrylamide/g acrylamide þ g bis-acrylamide)) (Ciavatta et al., 1996) or 2.6%C PAA slab gels (Bio-Rad Laboratories, USA), containing 2% of carrier ampholytes (CA’s). The CA’s used were composed of a mixture of 60% Ampholine (Pharmacia Biotech, Sweden) pH gradient from 3.5 to 5.0, 20% Ampholine with a pH gradient from 4.0 to 6.0 and 20% Ampholine with a pH gradient from 6.0 to 8.0 (Ciavatta et al., 1996). The slabs (260 110 0:5 mm) were prerun for 1, 2, 3 or 4 h in an electrophoretic cell (Multiphore II, LKB, Sweden),
L. Cavani et al. / Bioresource Technology 86 (2003) 45–52
47
Table 1 Characteristics of peat samples Samples
Origin
pHa
Standard (P s) 1 2 3 4 5 6 7 8
Ireland Italy Italy Italy Italy Germany Germany Russia
4.8 7.3 7.1 5.7 6.5 4.8 3.6 2.5
Commercial (P c) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Czech Rep. Ireland Ireland Ireland Ireland Ireland Italy Italy Italy Italy Italy Italy Norway Germany Germany Germany Russia Russia Russia Russia Ukraine
6.2 5.3 3.5 3.5 3.5 3.6 7.3 6.4 6.8 6.8 6.8 6.1 4.6 3.0 5.4 5.0 2.5 3.0 4.1 4.5 4.6
Moistureb (%)
Ashc (%)
TOC (%)
TEC (%)
HA þ FA (%)
4.0 10.6 4.5 8.5 8.9 9.1
8.8 74.3 65.9 15.3 57.4 2.7 0.8 6.2
52.3 12.4 20.7 41.9 21.5 49.3 49.2 47.2
26.3 7.6 12.8 26.1 16.1 21.0 21.5 31.0
23.6 5.9 10.4 22.1 13.6 18.2 17.3 25.8
8.9 10.2 9.6 8.6 10.2 10.3 5.1 7.1 9.8 6.4 7.1 17.3 8.5 12.0 6.9 9.6 8.2 9.6 8.6 11.6 9.3
12.2 1.9 8.3 2.0 1.3 2.3 64.7 60.0 42.4 44.1 58.3 53.3 6.3 1.3 14.6 8.2 7.2 8.4 7.6 19.2 21.5
45.1 50.4 47.4 50.6 49.9 47.6 20.9 20.5 26.5 28.1 21.3 20.7 51.7 42.3 43.7 45.7 48.1 41.8 51.2 40.6 39.5
25.3 39.8 24.6 31.7 32.1 17.9 11.4 14.9 18.2 19.6 13.6 14.6 34.2 15.0 21.7 21.2 34.9 21.6 26.4 30.2 23.7
22.1 27.8 21.6 28.4 29.1 14.8 9.6 12.3 16.5 18.0 11.0 12.2 29.6 11.0 19.0 19.1 31.8 16.8 25.7 26.5 21.0
7.1 3.2
a
Sample water ratio 1:10. Wet matter. c Dry matter. b
Table 2 Characteristics of leonardite samples Samples
Origin
pHa
Moistureb (%)
Ashc (%)
TOC (%)
TEC (%)
HA þ FA (%)
Standard (Le s) 1 2 3 4
USA USA USA USA
3.8 9.6 3.5 4.1
13.5 11.6 14.5 11.8
20.2 40.0 21.3 21.8
43.8 39.9 41.4 41.8
36.5 39.8 29.1 32.7
35.8 35.6 28.3 28.9
Commercial (Le c) 1 2 3 4 5 6 7
Spain Spain USA USA Unknown Unknown Unknown
2.9 5.5 3.7 3.8 4.6 3.6 5.1
7.7 11.2 12.8 18.4 11.6 5.7 9.8
60.8 42.7 16.0 21.0 20.4 72.5 29.7
21.2 31.2 45.4 39.1 48.2 15.7 40.4
14.6 24.4 34.9 32.0 43.6 10.9 25.1
12.2 22.6 33.5 30.6 42.0 9.9 22.6
a
Sample water ratio 1:10. Wet matter. c Dry matter. b
cooled at 2 C and supplied by a 2197 Power Supply (LKB, Sweden). The distance between the two electrodes was about 90 mm. The prerun and run conditions
were: max voltage 1200 V, power 0.9 W cm1 and current 0.6 mA cm1 . After loading an aliquot of sample (25–50 ll) the run was conducted for 2 h. After each run
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L. Cavani et al. / Bioresource Technology 86 (2003) 45–52
Table 3 Characteristics of humic extract of leonardite samples Samples
Origin
pHa
Moistureb (%)
Ashc (%)
TOC (%)
TEC (%)
HA þ FA (%)
Standard (HE s) 1 2 3 4
Spain USA USA USA
10.2 11.9 9.5 8.7
75.0 77.0 86.3 15.1
48.4 48.9 51.5 42.1
9.8 10.0 5.4 37.6
8.3 8.6 4.3 30.9
8.2 7.3 4.0 30.5
4.1 8.0 5.9 7.3 8.7 9.2 9.9 9.4 9.3 10.5 10.5 11.3 11.4 9.7 9.3 9.3
13.0 14.1 11.0 12.0 19.6 92.3 12.1 86.8 85.3 11.6 10.2 64.3 64.2 14.3 81.5 75.3
34.0 42.8 35.4 37.7 60.8 41.8 41.1 39.4 45.7 57.9 42.5 52.7 53.5 46.6 49.1 43.4
28.8 26.8 30.7 29.2 27.0 3.5 30.6 3.6 6.2 22.2 31.9 12.7 12.6 39.8 8.0 11.0
28.9 24.5 30.1 27.8 25.9 2.9 24.3 3.4 5.0 19.3 17.3 10.0 11.3 29.7 6.4 8.6
26.5 22.9 28.1 25.7 24.3 2.8 23.2 2.9 4.4 17.3 14.7 9.5 10.9 27.6 6.2 7.5
Commercial (HE c) 1 USA 2 USA 3 USA 4 USA 5 USA 6 USA 7 USA 8 USA 9 USA 10 USA 11 USA 12 USA 13 USA 14 Unknown 15 Unknown 16 Unknown a
Sample water ratio 1:10. Wet matter. c Dry matter. b
Table 4 Characteristics of lignite samples Samples
Origin
pHa
Moistureb (%)
Ashc (%)
TOC (%)
TEC (%)
HA þ FA (%)
Standard (Li) 1 2 3 4 5
Hungary Hungary Hungary Hungary USA
5.8 2.8 5.0 6.5 6.5
21.8 9.6 19.2 16.0 13.5
14.9 3.9 27.1 18.0 8.7
47.0 62.1 40.8 49.6 53.6
27.0 35.9 26.6 31.4 17.6
26.9 34.5 25.5 30.9 8.3
a
Sample water ratio 1:10. Wet matter. c Dry matter. b
the slabs were stained for two, under a gentle continuous stirring, in a solution containing 15% CH3 COOH (glacial, 100%), 15% CH3 CH2 OH (95%), 0.025% Blue Brilliant R-250 Coomassie (Merck, Germany) and 1% CuSO4 (99.5%). Then the slabs were treated overnight, under a gentle continuous stirring, with a solution containing: 15% CH3 COOH, 15% CH3 CH2 OH, 0.0025% Blue Brilliant R-250 Coomassie and 1% CuSO4 and finally with a solution prepared with 10% CH3 COOH and 10% CH3 CH2 OH to a complete destaining of the areas with no bands. After destaining the focused bands were scanned at 633 nm with an Ultroscan XL Enhanced Laser Densitometer (Pharmacia Biotech, Sweden). The bands of the EF densitograms were integrated and evaluated using the 2.1 version of the Gelscan software (Pharmacia Biotech, Sweden).
3. Results and discussion 3.1. Humification parameters The amounts of TOC, TEC and humificated carbon (HA þ FA) are reported in Tables 1–4. As with the results reported by Ciavatta et al. (1996) the TOC, TEC and HA þ FA differed both in standard and in commercial samples (Tables 1–4) and therefore could not be used to distinguish P from Le and Li samples. In the Figs. 1–3 are reported the distributions of calculated humification parameters. In spite of the heterogeneity of both matrices, due to the different origins and chemical properties (Tables 1–4), the humification parameters were able to characterise the different fertilisers. In fact, the differences between standard and
L. Cavani et al. / Bioresource Technology 86 (2003) 45–52
Fig. 1. Box plot of DH of fertiliser samples (P t: peats total, P s: peats standard, P c: peats commercial, Le t: leonardites total, Le s: leonardites standard, Le c: leonardites commercial, HE t: humic extracts of leonardite total, HE s: humic extracts of leonardite standard, HE c: humic extracts of leonardite commercial, Li: lignites).
49
Fig. 3. Box plot of HI of fertiliser samples (P t: peats total, P s: peats standard, P c: peats commercial, Le t: leonardites total, Le s: leonardites standard, Le c: leonardites commercial, HE t: humic extracts of leonardite total, HE s: humic extracts of leonardite standard, HE c: humic extracts of leonardite commercial, Li: lignites).
Nevertheless, the DH and the HI may only be used to distinguish P (DH < 90%, HI > 0:15) from Le and Li (Figs. 1 and 3), and may not be used to distinguish Le (DH > 90%; HI < 0:15) from Li (DH > 90%, HI < 0:12), because they show overlapping values. Because the HR found in P samples (Fig. 2) was lower than 65% while it was higher than 65% in Le samples it has been suggested (Ciavatta et al., 1996) that HR be used to distinguish P from Le samples. However, the mean HR of the Li samples (Fig. 2) differs significantly from those of the P samples for p ¼ 0:004 (Student t-test), but several samples of P showed an HR value similar to that of the Li samples (Fig. 2), and, finally, the number of Li samples was relatively limited (Table 4). On the basis of these results it is reasonable to suggest that HR may not be used for the identification of P from Le and Li, HR may only be used for identification of Le (HR > 65%). 3.2. Electrofocusing Fig. 2. Box plot of HR of fertiliser samples (P t: peats total, P s: peats standard, P c: peats commercial, Le t: leonardites total, Le s: leonardites standard, Le c: leonardites commercial, HE t: humic extracts of leonardite total, HE s: humic extracts of leonardite standard, HE c: humic extracts of leonardite commercial, Li: lignites).
commercial fertilisers were not significant for each type of fertiliser (Figs. 1–3), but the commercial products showed the most amount of data outside the 10th and 90th percentile.
In order to simplify the interpretation of the results obtained, the EF profiles have been subdivided into three main regions (Figs. 4–6): 1. region A, from pH 3.5 to about pH 3.8, characterised by a very intense band at a low apparent pI and by some other, not intense, bands; 2. region B, from pH 3.8 to pH 4.4, characterised by a group of four very well-resolved bands; 3. region C, with a group of bands focused over pH 4.4.
50
L. Cavani et al. / Bioresource Technology 86 (2003) 45–52
Fig. 4. Typical EF scan profiles at 633 nm of peat samples (P s: peats standard, P c: peats commercial), each sample is identified by a number detailed in Table 1.
Fig. 5. Typical EF scan profiles at 633 nm of leonardite and HE of leonardite samples (Le s: leonardite standard, Le c: leonardite commercial, HE: humic extract of leonardite), each sample is identified by a number detailed in Tables 2 and 3.
The EF profiles of P samples (Fig. 4) were characterised by two main groups of bands in the regions A and B, and sometimes by a third group of faint bands in the region near to the cathode (region C). Region B is the most important one, where it was possible to identify four bands (Fig. 4) with different intensities: bands 1–3 had a similar relative area, while band 4 had a bigger relative area. In some samples the area of band 4 was
Fig. 6. Typical EF scan profiles at 633 nm of lignite samples (Li: lignite), each sample is identified by a number detailed in Table 4.
similar to the areas of bands 1–3 (Fig. 4, low humified), while in some samples the area of band 4 was bigger than that of the bands 1–3 (Fig. 4, normally humified). The area of band 4 is very important because it corresponds to the humification level of P. In fact, several authors (De Nobili et al., 1990; Ciavatta and Govi, 1993; Kutsch and Schumacher, 1994) showed that the most complex and stable humic compounds are characterised by a pI higher than humic compounds with a lower humification level. Some of the samples that presented a big area in band 4 of the EF profile were also characterised by the presence of some bands in region C suggesting a higher humification level (Fig. 4, high humified). This result is apparently not in agreement with those reported by Ciavatta et al. (1996) where several P organic extracts showed a group of bands in this region. Ciavatta et al. (1996), however, used a 3.3%C PAA gel that was less porous than the 2.6%C PAA gel used in this work. It is reasonable to suppose that the organic compounds with higher molecular size were not able to run in the gel used by Ciavatta et al. (1996) and that they could not reach their focusing point. The EF profile of standard Le, commercial Le and HE of Le is shown in Fig. 5. The EF profiles of Le and HE are similar to P profiles in regions A and B, but in region C these profiles are characterised by a very intense group of bands (Fig. 5). This finding is in agreement with the hypothesis that the Le were composed of the most humified substances with an high apparent pI (pH > 4:4) (De Nobili et al., 1990). Band 4 of region B is more intense than bands 2 and 3 (Fig. 5), and the area of region C is 3–4 times bigger than the area of region B (Table 5). The EF profiles of HE samples are similar to
L. Cavani et al. / Bioresource Technology 86 (2003) 45–52 Table 5 Relative area (%) of bands from regions A, B and C calculated on the EF profiles at 633 nm Samples
n
Region A pH 3.5–3.8
Region B pH 3.8–4.4
Region C pH 4.4–6.0
P total P standard P commercial Le total Le standard Le commercial HE Le total HE Le standard HE Le commercial Li standard
29 8 21 11 4 7 20 4 16 5
51.7 9.5a 53.8 9.6 50.9 9.5 12.7 5.1 12.8 4.6 12.6 5.8 29.0 9.3 24.1 9.2 30.2 9.2 7.6 0.9
45.3 7.1 42.5 5.5 46.4 7.4 18.4 3.5 17.3 2.1 19.0 4.1 25.9 6.6 20.8 6.9 27.2 6.1 11.3 4.1
3.0 8.4 3.7 7.9 2.7 8.8 68.9 6.7 69.9 4.4 68.4 8.0 45.1 13.0 55.1 16.0 42.6 11.5 81.0 4.6
a
51
presence in region C of a very intense group of bands (relative area > 60%); • Li: HR ¼ 55–65%; absence or limited presence of bands 3–4 in region B; presence in region C of a poorly resolved group of bands (relative area > 75%). In conclusion, it is can by said that humification parameters (particularly HR) and EF are very useful for characterisation and identification of P, Le and Li in fertiliser samples.
Acknowledgements
Mean standard deviation.
EF profiles of Le samples (Fig. 5), the only difference is the relative area of region C; nearly 1.5–2 times bigger than the area of region B (Table 5). This reduction of materials with high apparent pI was probably due to the extraction processes. The EF profiles of Li samples (Fig. 6) was very different from those of P and Le, these profiles were characterised by a typical group of peaks with very low intensity in region B (only bands 3 and 4 were eventually present), and a poorly resolved group of bands in the region C: the integrated area of region C is 7–8 time greater than the area of region B (Table 5).
4. Conclusions Only partial characterisation of P, Le and Li is possible with the humification parameters: with HR is possible to distinguish Le from P and Li (Fig. 2), but not P from Li; with DH and HI it is possible to distinguish P from Le and Li (Figs. 1 and 3), but not Le from Li. The EF, instead, allows a precise characterisation of different fertilisers, and can distinguish between the different degrees of humification of P samples. On the base of the results shown here it is possible to propose the following classification: • P with a low humification level: HR < 40%, and/or absence of EF band 4 in region B; absence of bands in region C (relative area < 5%); • normally humified P: HR > 40%; presence of EF bands 1–4 in region B with similar intensity; absence of bands in region C (relative area < 5%); • very well humified P: HR > 50%; presence of EF bands 1–4. Bands 3 and 4 more intense than bands 1 and 2; bands in region C eventually present (relative area > 5%); • Le: HR > 65%; presence of EF bands 1–4 in region B; bands 1 and 4 more intense than bands 2 and 3;
This research was supported with funds provided by ‘‘Ispettorato Centrale Repressioni Frodi’’ of the Italian Ministry of the Agricultural and Forestry Politics (MiPAF). The authors thank Dr. Marco Govi for his help and valuable suggestions to develop this research.
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