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Evolution of organic matter in sewage sludge: A study based on the use of humification parameters and analytical electrofocusing
Bioresource Technology 44 (1993) 175-180
EVOLUTION OF ORGANIC MATTER IN SEWAGE SLUDGE: A STUDY BASED ON THE USE OF HUMIFICATION PARAMETERS A N D A N A L Y T I C A L ELECTROFOCUSING M. Govi, C. Ciavatta & C. Gessa Institute of Agricultural Chemistry, Universityof Bologna, Via S. Giacomo 7, 40126 Bologna, Italy (Received 24 August 1992; accepted 2 November 1992)
carbon of an organic extract, proposed the adoption of a new parameter of humification, the humification index (HI), to evaluate the extent of humification in organic materials. This method was modified by Ciavatta et al. (1990), who, in addition, proposed the use of two new parameters: the degree of humification (DH) and the humification rate (HR). These humification parameters have been applied with success to evaluate the humification of organic matter in soils (Govi et al., 1992), compost (De Nobili & Petrussi, 1988), and fertilizers (Ciavatta et aL, 1988) as well as to monitor the evolution of organic matter in pig slurries (Govi et al., 1989a). However, even though the humification parameters are very useful in determining the extent of humification, they give no information about the quality of the humic substances extracted. One of the most promising analytical techniques, which has been used by several authors to study humic substances extracted from soils (Govi et al., 1992), slurries (Govi et al., 1989b), composts (De Nobili et al., 1989), and, more recently, organic fertilizers (Govi et al., 1991a, b), is analytical electrofocusing (EF). This technique, based on the electrophoretic fractionation of the organic compounds in a predetermined pH gradient, has been shown to be a very useful tool to distinguish between a mature and a raw organic material as well as to monitor the changes in organic materials in pig slurries (Govi et al., 1989b). The objectives of the work reported in the present paper were: (a) by using the humification parameters, to monitor the evolution of the organic matter in sewage sludges from urban wastewater-treatment systems during aerobic and anaerobic maturation, and (b) by using EF, to evaluate the quality of the organic matter extracted from the sludges with three extractant solutions.
Abstract
The degree of humification (DH), humification index (HI), and humification rate (HR) were determined for seventeen samples of sewage sludges and composts from sewage sludges. The same samples, extracted with 0"5 M NaOH, 0"1 M Na4P207, and 0"1 M Na4P207 adjusted to p H 7, were analyzed with analytical electrofocusing (El:). The results clearly show that the organic matter changes much more quickly during composting than during the anearobic digestion. It was also found that the ability of the alkaline solutions to solubilize the organic complexes was greater the higher the apparent isoelectric point and the greater the reproducibilty of the analytical electrofocusing data. INTRODUCTION
Several problems are encountered in the agronomic use of sewage sludges from urban wastewater-treatment systems, including those due to high contents of heavy metals and the presence of xenobiotic compounds. However, one of the most important problems concerns the degree of maturation of the organic matter of the sludges. Fertilization with sewage sludges can be very risky if the organic matter has not been properly stabilized. Inadequate stabilization results in the presence of phytotoxic compounds or in their successive formation during the fermentation processes, which can be a cause of damage to crops. Many parameters have been proposed by several authors to monitor the changes in organic matter in composts, slurries, and sludges (Chanyasak & Kubota, 1981; Giusquiani et al., 1989; Herada & Inoko, 1980; Riffaldi et al., 1983; Riffaldi et al., 1986), but their application has not been reliable because these methods are often not useful for distinguishing between raw and mature materials. Sequi et al. (1986), using a new fractionation method to quantify the humifled (HA+FA) and the non-humified (NH) organic
METHODS
The samples of sewage sludges used (Table 1) were taken from the municipal wastewater-treatment plant of Faenza (Ravenna, Italy). The samples were dried at 65°C, crushed to pass a 0"25-mm sieve, and stored in plastic bottles for subsequent analysis.
Bioresource Technology 0960-8524/93/S06.00 © 1993 Elsevier Science Publishers Ltd, England. Printed in Great Britain 175
176
M. GovL C. Ciavatta, C. Gessa
Extraction and fractionation of the organic matter
A 10-g sample was shaken in a 150-ml centrifuge tube with 100 ml of 0"1 M NaOH plus 0.1 MNa4P207 for 3 h and centrifuged at 4000 g for 15 min. The resulting supernatant liquid was filtered at 0"8 /zm (Millipore, USA) and then divided into two fractions, the humified (HA+FA) and the non-humified (NH) fractions by using the method outlined in Fig. 1 and proposed by Ciavatta et al. (1990). The total organic carbon (TOC) in the solid samples was determined by a dichromate-oxidation method
(Ciavatta et al., 1989), and the total organic carbon in the liquid extracts was determined by using a similar method proposed by Ciavatta et al. (1990). Humification parameters
The amounts of organic carbon in the extracted (TEC), humified (HA+ FA), and non-humified (NH) fractions were used to determine the following three humification parameters: (i)
Humification Index (Sequi et al., 1986): NH H I = HP A " ' + ~ ;
Table 1. Samples of sewage sludges and composts used
Sludges D 1 samples a
D 2 samples a, b,
(ii)
sludge from an anaerobic digester sludge after anaerobic digester sludge partially dried and collected after 15-20 days of maturation
and b and c
D 3 samples a, b, c, and d
Composts C~A C2A
sludge mixed with straw sludge mixed with straw and composted in an aerated static pile for 40 days sludge mixed with straw and composted in an aerated static pile for 80 days sludge mixed with straw sludge mixed with straw and composted in a mixed pile for 40 days sludge mixed with straw and composted in a mixed pile for 80 days
C3A
CIR C2R C3R samples a, b, and c
Degree of Humification (Ciavatta et al., 1988, 1990): H A + FA DH% = - • 100; TEC
(iii) Humification Rate (Ciavatta et al., 1988, 1990): HA+FA HR% - - • 100. TOC
Electrofocusing (EF)
Extraction A 10-g sample was shaken in a 150-ml centrifuge tube with 100 ml of extractant solution for 3 h at 65°C and 100 r/rain in a Dubnoff water-bath. The following extractant solutions were used: 0"5 M NaOH, 0"1 M Na4P207 and 0"1 M Na4P207 adjusted to pH 7 with 85% H3PO 4. For simplification, later in this paper, these extracts are referred to as Na, PP, and PP7, respectively. After the extraction, the samples were centrifuged at 4000 g for 20 min; the supernantant liquids were filtered with a 0-8-#m Millipore filter and dialyzed in a MATRIX (extraction with alkali)
ORGANIC EXTRACT (H~SO, until pH <2
and centrifugation)
INSOLUBLE FRACTION: HUMIC ACID (HA)
SOLUBLE FRACTION: FULVlC ACID + NON-PHENOLIC SUBSTANCES
separation on PVP columns
(non-retained
fraction)
~.~
FULVIC ACID (FA)
(fraction retained and
re-eluted with 0.5M NaOH) NON-HUMIFIED FRACTION (NH)
Fig. 1.
HUMIFIED FRACTION (HA+FA)
Method of extraction and fractionation of the organic carbon from dried sludges and composts.
Organic matter changes in sewage sludge
177
Table 2. Some chemical characteristics of the samples of sludges and compost used
Materials
Nto,
NH~ -N
Ptot
TOC
TEC
HA + FA
NH
7.2 8"5
2.7 3.8
1.2 3.2
27.1 26.2
7.08 7.18
3.29 4.25
3.79 2"93
5"3 6"6 6.4
2.2 2.1 1.7
1.4 1.3 1.1
25.9 25.6 26.4
7.54 6"98 5.35
3"96 3.29 2.14
3"58 3.69 3.21
4.4 3.6 3"5 3"8
0.4 0.3 0"3 0"3
0"9 1.3 1.1 1.3
28"5 24.5 20.8 23.4
8"71 5.74 5.16 7.56
5"04 2.64 2"16 4.27
3"67 3.10 3"00 3"29
2"6
0"3
0"7
25"2
6"77
3"48
3'29
C2A
2"6
0"5
0"6
25"9
7"54
4"82
2"72
C3A
2"6
0"4
0"7
17-4
5"10
3"29
1"81
CIR
2"3
0"2
0"7
25"4
8"88
5'02
3"86
C2R
2"6
0'4
0"9
21"2
8"28
5"21
3"07
sample a C3R sample b C3R sample c
2.0 2.6 2.5
0'3 0.4 0.4
0"6 0.6 0.7
21.3 18.4 23.5
5.83 7.18 8.33
3"89 4.64 4.94
1.94 2.54 3"39
Sludges D 1sample a D 1sample b D 2 sample a D 2 sample b
D2 sample c D 3 sample a D 3 sample b
D 3 sample c D 3 sample d Composts CIA
C3R
TOC = total organic carbon; TEC =total extracted carbon; HA+ FA= carbon in the humic acid plus fulvic acid fraction; NH = carbon in the non-humified fraction. All data are expressed as percentages of the dry matter.
1000-daltons dialysis tube against distilled water. The 0"5 M NaOH extracts, before dialysis, were partially desalted with an ion-exchange resin (Amberlite IR 120 H + form) to reach a pH of about 9. All the dialyzed extracts (Table 2) were analyzed by the EF technique in order to obtain information about the nature of the organic carbon present. EF separation The electrofocusing (EF) analysis was conducted by using a 5.1% T, 3.7% C polyacrylamide-slab gel (T=(grams of acrylamide plus bisacrylamide)/100 ml of solution; C = grams of bisacrylamide/T%) containing 0"6% ampholytes with a predetermined pH gradient from 3-5 to 10 (Ampholine from Pharmacia -- LKB, Sweden) and 1"4% ampholytes (Ampholine) with a predetermined pH gradient from 4 to 6. The slabs (220x 110x0-8 mm) were prerun in a Multiphore II electrophoretic slab cell (Pharmacia -LKB) at I°C for 1 h and 15 min. Then, after the application of the samples near to the cathode, the slabs were run for 2 h and 30 min. The run conditions were preset with a 2177 power supply (LKB); the maximum values were 1200 V, 1"5 mA/cm, and 1 W/cm. After each run, the pH gradient was immediately measured with a specific surface electrode (Ingold, Switzerland), and the slabs were stained with a solution containing 0-5% Coomassie G-250, 10% ethanol, and
10% acetic acid for 3 h and destained with a solution containing 10% ethanol and 10% acetic acid. The focused bands were then scanned at 633 nm with an Ultroscan XL Enhanced Laser Densitometer (LKB), supported by the 2.1 version of the Gelscan software. RESULTS AND DISCUSSION As expected, the total organic carbon (TOC) tended to decrease with the ageing of the sludges and composts (Table 2). Generally, the humification index decreased during maturing of the organic matter of the composts, whereas the degree of humification increased (Table 3). This trend, however, was not found for the sludges, probably owing to the presence of a larger amount of pseudohumic substances that interfered during fractionation of the humic substances. The trend of the humification rate was not clearly defined. Although this parameter has been successfully used for the evaluation of the extent of humification in other materials, it does not appear to be a useful parameter to characterize the wastes studied in the present research. As expected, (Govi et al., 1989; De Nobili et al., 1989) in the EF profiles of the most-stabilized materials, there were more bands focused at the higher pH
178
M. Govi, C. Ciavatta, C. Gessa
Table 3. Index (HI), degree (DH), and rate (HR) of humification found in the samples of sludges and composts
Materials
HI
DH%
HR %
Sludges Dl sample a Dl sample b
1.15 0.69
46.5 59.2
12.1 16.2
D 2 sample a D 2 sample b D2 sample c
0"90 1.12 1"50
52.5 47.1 40.0
15"3 12"9 8.11
D3 sample a D3 sample b D 3 sample c D 3 sample d Composts
0.73 1.17 1.39 0"77
57.9 46.0 41.9 56.5
17-7 10.8 10.4 18.2
0.684
2
345
6789
0.000 1.155
2
345
6789
D2 0.0oo
CIA
0'95
51"4
13'8
C2A
0"56
63"9
18"6
C3A
0"55
64'5
18"9
C1R
0"77
56"5
19"7
C2R
0"59
62"9
24"6
1.a4,
C3R sample a C3R sample b C3R sample c
0"50 0"55 0.69
66.7 64.6 59"3
18.3 25.3 21.0
Fig. 2.
2
345
6789
D3 EF profiles of D~, D2, and D 3 extracted with PP7.
1,402
6789 345 values of the pH-gradient region. In contrast, the bands of the EF profiles of the raw or the poorly stabilized samples were focused mainly in the more acidic regions. This trend was found for all three of the extractant solutions used. In Fig. 2, the EF profiles of the PP7 extracts from samples D~, D2, and D 3 are shown. In the EF profile of the PP7 extract the D1, there is one evident band (1) focused at pH 3-5, one band (2) focused at pH 3"65, a group of bands (3, 4, and 5) focused from pH 3"8 to pH 3-9, and a group of bands (6, 7, 8, and 9) focused from pH 3-9 to pH 4.1. The same groups of bands appeared in the EF profiles of the D 2 and D 3 extracts, but, in the EF profile of D2, the bands focused at 3"65, the group of bands focused from pH 3"8 to pH 3"9, and band 6 are much more intense than those in the EF profile of D1. In the EF profile of the PP7 extracts from D3, bands 7, 8, and 9 are also more intense than the bands in the EF profiles of the D 1 extract. In Fig. 3 are shown the EF profiles of the PP7 extracts from CIA , C 2 A , and C3A. The three EF profiles are very similar in the region from pH 3"5 to pH 3"9: there is a band (1) focused at pH 3"5, another band (2) at pH 3.65, and a group of three bands (3, 4, and 5) focused from pH 3"8 to pH 3"9. In the EF profile of the C]A sample, there is a group of four wellresolved bands (6, 7, 8, and 9) in the pH gradient region from pH 3"9 to pH 4"1. In the same region, also in the EF profiles of the C2A and C3A extracts, there is a group of four well-focused bands, but bands 8 and 9 are more intense in the EF profile of C3A than in the
0.000
678 9
]IH
2
3,
~1011 / \ A ~ 12
CA
78
1.314
[
10 11 o.ooo~
Fig. 3.
~
--
C3A
EF profiles of CIA, C2A , and C3A extracted with PPT.
EF profile of C]A and C2A. In the EF profile of C2A , but particularly in the EF profile of the C3A extracts, there are three bands (10, 11, and 12) focused in the region from pH 4-2 to pH 4"4 that do not appear in the profile of C1A. In Fig. 4 are shown the EF profiles of the PP7 extracts from samples CaA and C3R. As also seen with
Organic matter changes in sewage sludge 0.989
345
C3R
6789
0"9Ii~961 I
179
345
6789
345
n789,u ~
O.OO( t 0.93."
• 678 CaA
I'
0.000 iJ
pp
6789
0.867
345 ~
Na
10
0.009[
Fig. 4.
EF profiles of C3R and C3A extracted with PP7-
the alkaline extracts (PP and Na), composting method A produced a more-matured material than composting method R. Indeed, in the EF profiles of C3A, bands 6, 7, 8, and 9, focused in the pH-gradient region from pH 3"9 to pH 4.1, are much more intense than in the EF profile of C3R. The choice of extractant does not appear to be important for recognition of the extent of maturation of a sludge or of a compost. It is, however, evident that the sludges and the composts of sludges exctracted with alkaline extractants are richer in compounds with higher apparent isoelectric points. Shown in Fig. 5 are the EF profiles of C2R extracted with PP7, PP and Na. The bands numbered 6, 7, 8, and 9 are focused in all three of the EF profiles, but, in the EF profile of the PP7 extract, the intensity of bands 7, 8, and 9 is lower than that in the EF profile of the PP extract. In the EF profile of the Na extract, band 9 is much more intense than in the other two profiles, whereas bands 1 and 2 are much less intense. The results obtained also show the good reproducibility of the method. In Fig. 6, for example, the EF profiles of the Na extract from the four D 3 samples are very similar, and the reproducibility is very good. On the basis of the hnmification parameters and the results of the EF analysis, the best treatment to achieve maturing of the sewage sludge was composting with straw and, in particular, technique A. The evolution of organic matter in the digester processes was found to be incomplete. The use of three classic extractants for humic substances made it possible to note that solubilization of the complexes with a higher apparent iso-
Fig. 5.
EF profiles of C2R extracted with PP7,PP, and Na.
1.243
O.OOO
2
3
2
3
0.850 c
O.O( 1.26~"
67
O.O00
9
0.557
1
45 / ~ d
0.000
Fig. 6.
EF profiles of D 3 samples a, b, c, and d extracted with Na.
electric point was better in the extracts obtained with more alkaline solutions. The good reproducibility of the EF data found in this study confirms the validity of this analytical method for the characterization of organic matter.
180
M. Govi, C. Ciavatta, C. Gessa
ACKNOWLEDGEMENTS T h e authors wish to thank the Centro Richerche Produzioni Animafi (C.R.P.A. of Reggio Emilia, Italy) for purchasing the samples used in this work. We also gratefully acknowledge Dr Giovanni Bonoretti for his technical assistance.
REFERENCES Chanyasak, V. & Kubota, H. (1981). Carbon, organic nitrogen ratio in water extract as measure of composting degradation. Journal of Fermentation Technology, 59, 215-19. Ciavatta, C., Vittori Antisari, L. & Sequi, P. (1988). A first approach to the characterization of the presence of humifled materials in organic fertilizers. Agrochimica, 32, 510-17. Ciavatta, C., Vittori Antisari, L. & Sequi, P. (1989). Determination of organic carbon in soils and fertilizers. Communications in Soil Science and Plant Analysis, 20,759-73. Ciavatta, C., Govi, M., Vittori Antisari, L. & Sequi, P. (1990a). Characterization of humified compounds by extraction and fractionation on solid polyvinylpyrrolidone. Journal of Chromatography, 509, 141-6. Ciavatta, C., Govi, M., Vittori Antisari, L. & Sequi, P. (1990b). Determination of organic carbon in aqueous extracts of soils and fertilizers. Communications in Soil Science and Plant Analysis, 22,795-807. De Nobili, M. & Petrussi, F. (1988). Humification Index (HI) as evaluation of the stabilization degree during composting. Journal of Fermentation Technology, 66, 577-82. De Nobili, M., Ciavatta, C. & Sequi, P. (1989). Evaluation of organic matter stabilization during composting by means of humification parameters and electrofocusing. In Proceedings of the International Symposium on Compost:
Production and Use. San Michele all'Adige, Italy, pp. 328-42. Giusquiani, P. L., Patumi, M. & Businelli, M. (1989). Chemical composition of fresh and composted urban waste. Plant and Soil 116,278-82. Govi, M., Ciavatta, C., Vittori Antisari, L. & Sequi, P. (1989a). Problemi relativi all'utilizzo d i effiuenti zootecnici in agricoltura. In Acque Reflue e Fanghi. ed. A. Frigerio CI-ESSE, Centro Scientifico Internazionale, Milan. pp. 422-31. Govi, M., Ciavatta, C., Vittori Antisari, L. & Sequi, P. (1989b). Characterization of organic materials by means of analytical electrofocusing. In Proceedings of the International Symposium on Humic Substances in the Aquatic and Terrestrial Environment. Link6ping, Sweden, pp. 143-9. Govi, M., Ciavatta, C., Vittori Antisari, L. & Sequi, P. (1991a). Characterization of humified substances in organic fertilizers by means of analytical electrofocusing (EF): a first approach. Fertilizer Research, 28,333-9. Govi, M., Montecchio, D. & Ciavatta, C. (1991b). Characterization of humic and humic-like substances in organic fertilizers and amendments. Finnish Humus News, 3, 303-8. 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 Science, 154, 8-13. Harada, Y. & Inoko, A. (1980). The measurement of the cation exchange capacity of composts for the estimation of the degree of maturity. Soil Science and Plant Nutrition, 26, 127-34. Riffaldi, R., Levi-Minzi, R. & Saviozzi, A. (1983). Humic fractions of organic wastes. Agriculture, Ecosystems and Environment, 10, 353-9. Riffaldi, R., Levi-Minzi, R., Pera, A. & de Bertoldi, M. (1986). Evaluation of compost maturity by means of chemical and microbial analyses. Waste Management and Research, 4, 387-96. Sequi, P., De Nobili, M., Leita, L. & Cercignani, G. (1986). A new index of humification. Agrochimica, 30, 175-9.
EVOLUTION OF ORGANIC MATTER IN SEWAGE SLUDGE: A STUDY BASED ON THE USE OF HUMIFICATION PARAMETERS A N D A N A L Y T I C A L ELECTROFOCUSING M. Govi, C. Ciavatta & C. Gessa Institute of Agricultural Chemistry, Universityof Bologna, Via S. Giacomo 7, 40126 Bologna, Italy (Received 24 August 1992; accepted 2 November 1992)
carbon of an organic extract, proposed the adoption of a new parameter of humification, the humification index (HI), to evaluate the extent of humification in organic materials. This method was modified by Ciavatta et al. (1990), who, in addition, proposed the use of two new parameters: the degree of humification (DH) and the humification rate (HR). These humification parameters have been applied with success to evaluate the humification of organic matter in soils (Govi et al., 1992), compost (De Nobili & Petrussi, 1988), and fertilizers (Ciavatta et aL, 1988) as well as to monitor the evolution of organic matter in pig slurries (Govi et al., 1989a). However, even though the humification parameters are very useful in determining the extent of humification, they give no information about the quality of the humic substances extracted. One of the most promising analytical techniques, which has been used by several authors to study humic substances extracted from soils (Govi et al., 1992), slurries (Govi et al., 1989b), composts (De Nobili et al., 1989), and, more recently, organic fertilizers (Govi et al., 1991a, b), is analytical electrofocusing (EF). This technique, based on the electrophoretic fractionation of the organic compounds in a predetermined pH gradient, has been shown to be a very useful tool to distinguish between a mature and a raw organic material as well as to monitor the changes in organic materials in pig slurries (Govi et al., 1989b). The objectives of the work reported in the present paper were: (a) by using the humification parameters, to monitor the evolution of the organic matter in sewage sludges from urban wastewater-treatment systems during aerobic and anaerobic maturation, and (b) by using EF, to evaluate the quality of the organic matter extracted from the sludges with three extractant solutions.
Abstract
The degree of humification (DH), humification index (HI), and humification rate (HR) were determined for seventeen samples of sewage sludges and composts from sewage sludges. The same samples, extracted with 0"5 M NaOH, 0"1 M Na4P207, and 0"1 M Na4P207 adjusted to p H 7, were analyzed with analytical electrofocusing (El:). The results clearly show that the organic matter changes much more quickly during composting than during the anearobic digestion. It was also found that the ability of the alkaline solutions to solubilize the organic complexes was greater the higher the apparent isoelectric point and the greater the reproducibilty of the analytical electrofocusing data. INTRODUCTION
Several problems are encountered in the agronomic use of sewage sludges from urban wastewater-treatment systems, including those due to high contents of heavy metals and the presence of xenobiotic compounds. However, one of the most important problems concerns the degree of maturation of the organic matter of the sludges. Fertilization with sewage sludges can be very risky if the organic matter has not been properly stabilized. Inadequate stabilization results in the presence of phytotoxic compounds or in their successive formation during the fermentation processes, which can be a cause of damage to crops. Many parameters have been proposed by several authors to monitor the changes in organic matter in composts, slurries, and sludges (Chanyasak & Kubota, 1981; Giusquiani et al., 1989; Herada & Inoko, 1980; Riffaldi et al., 1983; Riffaldi et al., 1986), but their application has not been reliable because these methods are often not useful for distinguishing between raw and mature materials. Sequi et al. (1986), using a new fractionation method to quantify the humifled (HA+FA) and the non-humified (NH) organic
METHODS
The samples of sewage sludges used (Table 1) were taken from the municipal wastewater-treatment plant of Faenza (Ravenna, Italy). The samples were dried at 65°C, crushed to pass a 0"25-mm sieve, and stored in plastic bottles for subsequent analysis.
Bioresource Technology 0960-8524/93/S06.00 © 1993 Elsevier Science Publishers Ltd, England. Printed in Great Britain 175
176
M. GovL C. Ciavatta, C. Gessa
Extraction and fractionation of the organic matter
A 10-g sample was shaken in a 150-ml centrifuge tube with 100 ml of 0"1 M NaOH plus 0.1 MNa4P207 for 3 h and centrifuged at 4000 g for 15 min. The resulting supernatant liquid was filtered at 0"8 /zm (Millipore, USA) and then divided into two fractions, the humified (HA+FA) and the non-humified (NH) fractions by using the method outlined in Fig. 1 and proposed by Ciavatta et al. (1990). The total organic carbon (TOC) in the solid samples was determined by a dichromate-oxidation method
(Ciavatta et al., 1989), and the total organic carbon in the liquid extracts was determined by using a similar method proposed by Ciavatta et al. (1990). Humification parameters
The amounts of organic carbon in the extracted (TEC), humified (HA+ FA), and non-humified (NH) fractions were used to determine the following three humification parameters: (i)
Humification Index (Sequi et al., 1986): NH H I = HP A " ' + ~ ;
Table 1. Samples of sewage sludges and composts used
Sludges D 1 samples a
D 2 samples a, b,
(ii)
sludge from an anaerobic digester sludge after anaerobic digester sludge partially dried and collected after 15-20 days of maturation
and b and c
D 3 samples a, b, c, and d
Composts C~A C2A
sludge mixed with straw sludge mixed with straw and composted in an aerated static pile for 40 days sludge mixed with straw and composted in an aerated static pile for 80 days sludge mixed with straw sludge mixed with straw and composted in a mixed pile for 40 days sludge mixed with straw and composted in a mixed pile for 80 days
C3A
CIR C2R C3R samples a, b, and c
Degree of Humification (Ciavatta et al., 1988, 1990): H A + FA DH% = - • 100; TEC
(iii) Humification Rate (Ciavatta et al., 1988, 1990): HA+FA HR% - - • 100. TOC
Electrofocusing (EF)
Extraction A 10-g sample was shaken in a 150-ml centrifuge tube with 100 ml of extractant solution for 3 h at 65°C and 100 r/rain in a Dubnoff water-bath. The following extractant solutions were used: 0"5 M NaOH, 0"1 M Na4P207 and 0"1 M Na4P207 adjusted to pH 7 with 85% H3PO 4. For simplification, later in this paper, these extracts are referred to as Na, PP, and PP7, respectively. After the extraction, the samples were centrifuged at 4000 g for 20 min; the supernantant liquids were filtered with a 0-8-#m Millipore filter and dialyzed in a MATRIX (extraction with alkali)
ORGANIC EXTRACT (H~SO, until pH <2
and centrifugation)
INSOLUBLE FRACTION: HUMIC ACID (HA)
SOLUBLE FRACTION: FULVlC ACID + NON-PHENOLIC SUBSTANCES
separation on PVP columns
(non-retained
fraction)
~.~
FULVIC ACID (FA)
(fraction retained and
re-eluted with 0.5M NaOH) NON-HUMIFIED FRACTION (NH)
Fig. 1.
HUMIFIED FRACTION (HA+FA)
Method of extraction and fractionation of the organic carbon from dried sludges and composts.
Organic matter changes in sewage sludge
177
Table 2. Some chemical characteristics of the samples of sludges and compost used
Materials
Nto,
NH~ -N
Ptot
TOC
TEC
HA + FA
NH
7.2 8"5
2.7 3.8
1.2 3.2
27.1 26.2
7.08 7.18
3.29 4.25
3.79 2"93
5"3 6"6 6.4
2.2 2.1 1.7
1.4 1.3 1.1
25.9 25.6 26.4
7.54 6"98 5.35
3"96 3.29 2.14
3"58 3.69 3.21
4.4 3.6 3"5 3"8
0.4 0.3 0"3 0"3
0"9 1.3 1.1 1.3
28"5 24.5 20.8 23.4
8"71 5.74 5.16 7.56
5"04 2.64 2"16 4.27
3"67 3.10 3"00 3"29
2"6
0"3
0"7
25"2
6"77
3"48
3'29
C2A
2"6
0"5
0"6
25"9
7"54
4"82
2"72
C3A
2"6
0"4
0"7
17-4
5"10
3"29
1"81
CIR
2"3
0"2
0"7
25"4
8"88
5'02
3"86
C2R
2"6
0'4
0"9
21"2
8"28
5"21
3"07
sample a C3R sample b C3R sample c
2.0 2.6 2.5
0'3 0.4 0.4
0"6 0.6 0.7
21.3 18.4 23.5
5.83 7.18 8.33
3"89 4.64 4.94
1.94 2.54 3"39
Sludges D 1sample a D 1sample b D 2 sample a D 2 sample b
D2 sample c D 3 sample a D 3 sample b
D 3 sample c D 3 sample d Composts CIA
C3R
TOC = total organic carbon; TEC =total extracted carbon; HA+ FA= carbon in the humic acid plus fulvic acid fraction; NH = carbon in the non-humified fraction. All data are expressed as percentages of the dry matter.
1000-daltons dialysis tube against distilled water. The 0"5 M NaOH extracts, before dialysis, were partially desalted with an ion-exchange resin (Amberlite IR 120 H + form) to reach a pH of about 9. All the dialyzed extracts (Table 2) were analyzed by the EF technique in order to obtain information about the nature of the organic carbon present. EF separation The electrofocusing (EF) analysis was conducted by using a 5.1% T, 3.7% C polyacrylamide-slab gel (T=(grams of acrylamide plus bisacrylamide)/100 ml of solution; C = grams of bisacrylamide/T%) containing 0"6% ampholytes with a predetermined pH gradient from 3-5 to 10 (Ampholine from Pharmacia -- LKB, Sweden) and 1"4% ampholytes (Ampholine) with a predetermined pH gradient from 4 to 6. The slabs (220x 110x0-8 mm) were prerun in a Multiphore II electrophoretic slab cell (Pharmacia -LKB) at I°C for 1 h and 15 min. Then, after the application of the samples near to the cathode, the slabs were run for 2 h and 30 min. The run conditions were preset with a 2177 power supply (LKB); the maximum values were 1200 V, 1"5 mA/cm, and 1 W/cm. After each run, the pH gradient was immediately measured with a specific surface electrode (Ingold, Switzerland), and the slabs were stained with a solution containing 0-5% Coomassie G-250, 10% ethanol, and
10% acetic acid for 3 h and destained with a solution containing 10% ethanol and 10% acetic acid. The focused bands were then scanned at 633 nm with an Ultroscan XL Enhanced Laser Densitometer (LKB), supported by the 2.1 version of the Gelscan software. RESULTS AND DISCUSSION As expected, the total organic carbon (TOC) tended to decrease with the ageing of the sludges and composts (Table 2). Generally, the humification index decreased during maturing of the organic matter of the composts, whereas the degree of humification increased (Table 3). This trend, however, was not found for the sludges, probably owing to the presence of a larger amount of pseudohumic substances that interfered during fractionation of the humic substances. The trend of the humification rate was not clearly defined. Although this parameter has been successfully used for the evaluation of the extent of humification in other materials, it does not appear to be a useful parameter to characterize the wastes studied in the present research. As expected, (Govi et al., 1989; De Nobili et al., 1989) in the EF profiles of the most-stabilized materials, there were more bands focused at the higher pH
178
M. Govi, C. Ciavatta, C. Gessa
Table 3. Index (HI), degree (DH), and rate (HR) of humification found in the samples of sludges and composts
Materials
HI
DH%
HR %
Sludges Dl sample a Dl sample b
1.15 0.69
46.5 59.2
12.1 16.2
D 2 sample a D 2 sample b D2 sample c
0"90 1.12 1"50
52.5 47.1 40.0
15"3 12"9 8.11
D3 sample a D3 sample b D 3 sample c D 3 sample d Composts
0.73 1.17 1.39 0"77
57.9 46.0 41.9 56.5
17-7 10.8 10.4 18.2
0.684
2
345
6789
0.000 1.155
2
345
6789
D2 0.0oo
CIA
0'95
51"4
13'8
C2A
0"56
63"9
18"6
C3A
0"55
64'5
18"9
C1R
0"77
56"5
19"7
C2R
0"59
62"9
24"6
1.a4,
C3R sample a C3R sample b C3R sample c
0"50 0"55 0.69
66.7 64.6 59"3
18.3 25.3 21.0
Fig. 2.
2
345
6789
D3 EF profiles of D~, D2, and D 3 extracted with PP7.
1,402
6789 345 values of the pH-gradient region. In contrast, the bands of the EF profiles of the raw or the poorly stabilized samples were focused mainly in the more acidic regions. This trend was found for all three of the extractant solutions used. In Fig. 2, the EF profiles of the PP7 extracts from samples D~, D2, and D 3 are shown. In the EF profile of the PP7 extract the D1, there is one evident band (1) focused at pH 3-5, one band (2) focused at pH 3"65, a group of bands (3, 4, and 5) focused from pH 3"8 to pH 3-9, and a group of bands (6, 7, 8, and 9) focused from pH 3-9 to pH 4.1. The same groups of bands appeared in the EF profiles of the D 2 and D 3 extracts, but, in the EF profile of D2, the bands focused at 3"65, the group of bands focused from pH 3"8 to pH 3"9, and band 6 are much more intense than those in the EF profile of D1. In the EF profile of the PP7 extracts from D3, bands 7, 8, and 9 are also more intense than the bands in the EF profiles of the D 1 extract. In Fig. 3 are shown the EF profiles of the PP7 extracts from CIA , C 2 A , and C3A. The three EF profiles are very similar in the region from pH 3"5 to pH 3"9: there is a band (1) focused at pH 3"5, another band (2) at pH 3.65, and a group of three bands (3, 4, and 5) focused from pH 3"8 to pH 3"9. In the EF profile of the C]A sample, there is a group of four wellresolved bands (6, 7, 8, and 9) in the pH gradient region from pH 3"9 to pH 4"1. In the same region, also in the EF profiles of the C2A and C3A extracts, there is a group of four well-focused bands, but bands 8 and 9 are more intense in the EF profile of C3A than in the
0.000
678 9
]IH
2
3,
~1011 / \ A ~ 12
CA
78
1.314
[
10 11 o.ooo~
Fig. 3.
~
--
C3A
EF profiles of CIA, C2A , and C3A extracted with PPT.
EF profile of C]A and C2A. In the EF profile of C2A , but particularly in the EF profile of the C3A extracts, there are three bands (10, 11, and 12) focused in the region from pH 4-2 to pH 4"4 that do not appear in the profile of C1A. In Fig. 4 are shown the EF profiles of the PP7 extracts from samples CaA and C3R. As also seen with
Organic matter changes in sewage sludge 0.989
345
C3R
6789
0"9Ii~961 I
179
345
6789
345
n789,u ~
O.OO( t 0.93."
• 678 CaA
I'
0.000 iJ
pp
6789
0.867
345 ~
Na
10
0.009[
Fig. 4.
EF profiles of C3R and C3A extracted with PP7-
the alkaline extracts (PP and Na), composting method A produced a more-matured material than composting method R. Indeed, in the EF profiles of C3A, bands 6, 7, 8, and 9, focused in the pH-gradient region from pH 3"9 to pH 4.1, are much more intense than in the EF profile of C3R. The choice of extractant does not appear to be important for recognition of the extent of maturation of a sludge or of a compost. It is, however, evident that the sludges and the composts of sludges exctracted with alkaline extractants are richer in compounds with higher apparent isoelectric points. Shown in Fig. 5 are the EF profiles of C2R extracted with PP7, PP and Na. The bands numbered 6, 7, 8, and 9 are focused in all three of the EF profiles, but, in the EF profile of the PP7 extract, the intensity of bands 7, 8, and 9 is lower than that in the EF profile of the PP extract. In the EF profile of the Na extract, band 9 is much more intense than in the other two profiles, whereas bands 1 and 2 are much less intense. The results obtained also show the good reproducibility of the method. In Fig. 6, for example, the EF profiles of the Na extract from the four D 3 samples are very similar, and the reproducibility is very good. On the basis of the hnmification parameters and the results of the EF analysis, the best treatment to achieve maturing of the sewage sludge was composting with straw and, in particular, technique A. The evolution of organic matter in the digester processes was found to be incomplete. The use of three classic extractants for humic substances made it possible to note that solubilization of the complexes with a higher apparent iso-
Fig. 5.
EF profiles of C2R extracted with PP7,PP, and Na.
1.243
O.OOO
2
3
2
3
0.850 c
O.O( 1.26~"
67
O.O00
9
0.557
1
45 / ~ d
0.000
Fig. 6.
EF profiles of D 3 samples a, b, c, and d extracted with Na.
electric point was better in the extracts obtained with more alkaline solutions. The good reproducibility of the EF data found in this study confirms the validity of this analytical method for the characterization of organic matter.
180
M. Govi, C. Ciavatta, C. Gessa
ACKNOWLEDGEMENTS T h e authors wish to thank the Centro Richerche Produzioni Animafi (C.R.P.A. of Reggio Emilia, Italy) for purchasing the samples used in this work. We also gratefully acknowledge Dr Giovanni Bonoretti for his technical assistance.
REFERENCES Chanyasak, V. & Kubota, H. (1981). Carbon, organic nitrogen ratio in water extract as measure of composting degradation. Journal of Fermentation Technology, 59, 215-19. Ciavatta, C., Vittori Antisari, L. & Sequi, P. (1988). A first approach to the characterization of the presence of humifled materials in organic fertilizers. Agrochimica, 32, 510-17. Ciavatta, C., Vittori Antisari, L. & Sequi, P. (1989). Determination of organic carbon in soils and fertilizers. Communications in Soil Science and Plant Analysis, 20,759-73. Ciavatta, C., Govi, M., Vittori Antisari, L. & Sequi, P. (1990a). Characterization of humified compounds by extraction and fractionation on solid polyvinylpyrrolidone. Journal of Chromatography, 509, 141-6. Ciavatta, C., Govi, M., Vittori Antisari, L. & Sequi, P. (1990b). Determination of organic carbon in aqueous extracts of soils and fertilizers. Communications in Soil Science and Plant Analysis, 22,795-807. De Nobili, M. & Petrussi, F. (1988). Humification Index (HI) as evaluation of the stabilization degree during composting. Journal of Fermentation Technology, 66, 577-82. De Nobili, M., Ciavatta, C. & Sequi, P. (1989). Evaluation of organic matter stabilization during composting by means of humification parameters and electrofocusing. In Proceedings of the International Symposium on Compost:
Production and Use. San Michele all'Adige, Italy, pp. 328-42. Giusquiani, P. L., Patumi, M. & Businelli, M. (1989). Chemical composition of fresh and composted urban waste. Plant and Soil 116,278-82. Govi, M., Ciavatta, C., Vittori Antisari, L. & Sequi, P. (1989a). Problemi relativi all'utilizzo d i effiuenti zootecnici in agricoltura. In Acque Reflue e Fanghi. ed. A. Frigerio CI-ESSE, Centro Scientifico Internazionale, Milan. pp. 422-31. Govi, M., Ciavatta, C., Vittori Antisari, L. & Sequi, P. (1989b). Characterization of organic materials by means of analytical electrofocusing. In Proceedings of the International Symposium on Humic Substances in the Aquatic and Terrestrial Environment. Link6ping, Sweden, pp. 143-9. Govi, M., Ciavatta, C., Vittori Antisari, L. & Sequi, P. (1991a). Characterization of humified substances in organic fertilizers by means of analytical electrofocusing (EF): a first approach. Fertilizer Research, 28,333-9. Govi, M., Montecchio, D. & Ciavatta, C. (1991b). Characterization of humic and humic-like substances in organic fertilizers and amendments. Finnish Humus News, 3, 303-8. 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 Science, 154, 8-13. Harada, Y. & Inoko, A. (1980). The measurement of the cation exchange capacity of composts for the estimation of the degree of maturity. Soil Science and Plant Nutrition, 26, 127-34. Riffaldi, R., Levi-Minzi, R. & Saviozzi, A. (1983). Humic fractions of organic wastes. Agriculture, Ecosystems and Environment, 10, 353-9. Riffaldi, R., Levi-Minzi, R., Pera, A. & de Bertoldi, M. (1986). Evaluation of compost maturity by means of chemical and microbial analyses. Waste Management and Research, 4, 387-96. Sequi, P., De Nobili, M., Leita, L. & Cercignani, G. (1986). A new index of humification. Agrochimica, 30, 175-9.