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Chemical characterization of humic substances extracted from organic-waste-amended soils
Bioresource Technology 40 (1992) 275-282
Chemical Characterization of Humic Substances Extracted from Organic-Waste-Amended Softs A. Piccolo Istituto Sperimentale per 1o Studio e la Difesa del Suolo, Piazza M. D'Azeglio 30, Firenze, Italy
E Zaccheo & P. G. Genevini Istituto Chimica Agraria, Universitfi di Milano, Via della Celoria, Milano, Italy (Received 24 October 1990; accepted 18 June 1991)
Abstract
Humic substances were extracted from a soil treated, in a 4-year experiment, at different rates with a sludge from anaerobic treatment of combined civil and industrial wastes, and with agricultural manure. Elemental and chemical analyses, molecular weight (MW) distribution and infrared (IR) spectroscopy were performed on the purified humic acids (HA). Organic wastes significantly increased the HA content of the treated soils and improved CEC. The C/N, C/H and C/O ratios of HA extracted from the original wastes showed a higher degree of humification for sludge than for manure. This difference was also noticed for the C/N ratio of soil humic extracts, indicating a faster humification process for the sludges in soil. The content of oxygen-containing functional groups was lower than the 'model' HA reported in the literature, and even more so for HA from sludges, reflecting their anaerobic formation. M W distribution and E4/E 6 ratios showed that the sludge material had a higher molecular complexity than manure, supporting the high degree of humification attributed to the former. HA extracted from sludgetreated soils revealed a molecular dimension increasing with the application doses of waste material. Infrared spectra showed that HA formed in soils after waste additions reflected the chemical composition of the original organic material, which was rich in aliphatic groups and peptides for sludge and in carbohydrates for manure. On the basis of this study, it is concluded that not only are organic waste additions able to build up the HA content in soils but the HA formed assume the chemical characteristics and the degree of humification of the original material.
Key words: Humus, soil, organic waste.
INTRODUCTION The worldwide growing demand for means of disposing organic wastes derived from agricultural productions and urban and industrial refuses has increasingly turned to soils as a highly suitable acceptor. It is claimed that the soil chemical and biological activity may efficiently and safely incorporate organic wastes into the soil organic components. This practice often results in an improvement in the chemical and physical properties of the soil (Marchesini et al., 1988; Mbagwu & Piccolo, 1989, 1990), although there is an increasing awareness of the risk of groundwater contamination by pollutants such as heavy metals leached down in soluble organic matter complexes (Campanella et al., 1989). The realization that organic matter introduced into soils by waste additions may alter the soil capacity to prevent pollutants from a downward mobility has led to increased research on organic matter characterization, since information on humus chemical properties may be useful to predict the fate of a pollutant in the soil environment. Sugahara & Inoko (1981) reported on humic acid properties of a city refuse compost, Petronio et al. (1989) proposed a fractionation scheme of the organic components of urban and industrial sludges for a better evaluation of the chemical properties, Petrussi et al. (1988) investigated the characteristics of organic matter from animal manures after digestion by earthworms, Inbar et aL (1990) followed the chemical changes of humus during the compost-
275 Bioresource Technology 0960-8524/92/S05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain
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A. Piccolo, P. Zaccheo, P. G. Genevini
ing of solid wastes from wineries. Other works studied the strength of metal complexes with soluble fulvic acids extracted from anaerobically digested sewage sludges (Senesi & Sposito, 1984). Less work has been done on the chemical characteristics of soil humic substances extracted after addition of organic wastes to soils. Piccolo & Mbagwu (1990) reported that addition of pig and cattle slurry and sewage sludge to soils in a long term experiment resulted in a selective increase of the high-molecular-weight humic fraction in soil micro-aggregates. The objective of this work was to investigate the general chemical and physical-chemical characteristics of humic substances extracted from soils treated with anaerobically digested sludge and cattle manure.
METHODS Soil and organic wastes A sandy loam soil classified as Fluventic Xerochrept was sampled in an agricultural area south of Milano, Italy. The anaerobic sludge (S) came from a civil and industrial waste water treatment plant, whereas cattle manure (M) originated from a livestock breeding farm. Some of the chemical characteristics of the soil, sludge and manure are reported in Table 1. Analyses were performed according to ASA Methods of Soil Analysis (1982). The sampled soil was used to fill greenhouse pots set for a 4-year experiment with the following amendments and rates (dry matter): control pot with only mineral fertilization (soil); 25 t hayear-t of sludge (SS1); 50 t ha-1 year- ~of sludge (SS2); 100 t ha-~ year-~ of sludge (SS3); 25 t ha-J year-J of cattle manure (SM1). Four replicates were arranged for each treatment. During the experimental period, Lolium italieum, for a total amount of 16 cuts, was grown in each pot. At the end of the experiment, a soil sample (0-15 cm)for each replicate of each treatment was collected. These were thoroughly mixed and four subsamples for each treatment were obtained, air dried, sieved (2 mm) and stored in the freezer for subsequent analysis. Extraction of humic acids Soil samples were suspended in 0.05 M HC1 for 24 h, filtered, washed with deionized H20 until chloride-free and then air dried. The samples were ground and suspended in 0.5 M NaOH
(soil:solution, 1:5) under a n N 2 atmosphere for 24 h. The alkaline extract was then centrifuged at 3000 g for 30 min, the precipitate was acidified with HC1 to pH 2, decanted at room temperature for 24 h and centrifuged to separate humic from fulvic acids (FA). The humic acids (HA) were then redissolved in 0.1 N NaOH under N2 and reprecipitated with 0-5 N HC1. The HA were further purified by a 24-h treatment with a 0.5% HCI/HF mixture with stirring. This treatment was repeated twice to lower the humic acid ash content. Purified HA were transferred to Visking dialysis tubes with an exclusion limit of 8000-15000 Dalton, and dialysed against deionized water for 5 days, and then freeze-dried. The extraction yield averaged approximately 4 g kg- ~of soil. Chemical characterization The C, H, N, S, elemental composition of HA was determined on A N A 1500 Carlo Erba instrumentation. Total acidity was determined by the Ba(OH)2 method, and values for carboxylic groups were obtained through the Ca-acetate procedure (Wright & Schnitzer, 1959). Phenolic groups were calculated by subtracting carboxylic groups from total acidity. Ketonic groups were determined by the hydroxylamine hydrochloride method (Fritz et al., 1959) and methoxyls by the Zeisel method (Piccolo, 1984); E 4 / E 6 w a s the ratio of absorbances at 465 nm and 665 nm (Chen etal., 1977). Chemical characterization was repeated twice for each sub-sample, reaching a total of 8 data/ treatment. The data obtained were statistically computed with ANOVA and Tuckey's alternate test.
Table 1. Analytical parameters for soil, sludge (S), and manure (M)
pH OM (%) N Tot. (%) P Tot. (%) CEC (meq/100) HA (%) AI (ppm) Cd (ppm) Cu (ppm) Cr (ppm) Ni (ppm) Pb (ppm) Zn (ppm)
Soil
S
M
7.5 2-06 0.13 0.12 16.7 0.32 Tr. Tr. Tr. Tr. Tr. Tr. Tr.
7.1 36,2 1,4 3,8 ND 1-32 2.01 47.2 724 1489 494 586 3410
9.06 72.4 2.67 1.4 ND 12-7 Tr. Tr. Tr. Tr. Tr. Tr. Tr.
ND, not determined;Tr., traces.
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Humic substances from waste-amended soils
Molecular weight distribution Molecular weight distribution of HA was determined using Sephacryl $200 as a molecular sieve in the gel permeation chromatography technique. This matrix, which was packed in a 70 cm x 1.5 cm Pharmacia column, has an upper exclusion limit of 2 5 0 0 0 0 daltons and, being a Dextran further crosslinked with N,N'-methylene bisacrylamide, Sephacryl has a higher rigidity than Sephadex, and allows higher flow rates and better control of pore size. The gel packing solution and the eluent was a 1 M 2-amino-2-hydroxymethyl1,3-propanediol (TRIS) buffered at pH 9 (Swift & Posner, 1971 ). Elution pressure was assured by a peristaltic pump giving a flow rate of 6-8 mi h Samples for chromatography (20 mg) diluted in 1 ml of TRIS were inserted on to the column surface by a four-way injection valve (Pharmacia SRV 4). The absorbance of the eluate was measured at 470 nm and the subsequent chromatograms were automatically produced by a continuous flow detector ISCO UA-5 with an incorporated recorder. The void volume of the column (V0) was assessed with Blue Dextran 2000. Other details on the column and gel calibration are reported elsewhere (Piccolo & Mirabella, 1987). Infrared spectra Infrared spectra were recorded using a Perkin Elmer 882 double-beam spectrometer. Samples for infrared analysis were prepared by mixing 1 mg of freeze-dried humic material with 300 mg of pre-dried and pulverized spectroscopic grade KBr. The KBr pellets were prepared as outlined by Piccolo & Stevenson (1982).
RESULTS AND DISCUSSION Extractable HA and soil CEC Yields of H A and the CEC for each soil at the end of the experiment are reported in Table 2. T h e percentage of extractable H A in the control soil decreased noticeably after 4 years, whereas soil samples treated with both manure and different amounts of sludge showed increased content of HA. Addition of sludges produced a H A content enhancement parallel to the application doses, which reached 200% of control for the highest sludge addition. Manure treatment increased soil H A content to a level not significantly different from the one corresponding to the equivalent amount of sludges. Also the CEC seemed to be
Table 2. Percentageof soil HA and soil CEC
Soil SM1 SS1 SS2 SS3
HA (%)
CEC ¢rneq/lO0)
0.21 a" 0'38ab 0-36 ab 0.42 b 0-66 c
18-8 a 21.2 a 19.7 a 21.6 a 25.0 b
"Means in the same column followed by the same letter are not significantlydifferent (P = 0"05, n = 8).
positively affected by the sludge and manure treatments, although its increase was significantly different from the control only at the highest level of sludge addition. These results indicate that organic wastes effectively build up the H A content of soils and that they have a beneficial effect on a soil-fertility parameter such as CEC. Elemental composition and functional group analysis of HA H A extracted from soils commonly have similar elemental compositions regardless of climatic and soil conditions (Schnitzer, 1978). The elemental composition and atomic ratios of H A extracted from the treated soils as well as that of 'model' H A and FA are presented in Table 3. Notwithstanding the type of organic waste and applied doses all the parameters found were strikingly similar. It is noteworthy, however, that the average composition of the HA extracted from the treated soils resembles more closely that of the 'model' FA than that of the 'model' HA. Both 'model' H A and FA represented a large number of humic materials formed under widely differing conditions (Schnitzer, 1978). Though the C and H contents of H A from the control soil, the soil plus manure, and soil plus different sludge doses, are close to the values reported for the 'model' FA, the N and S contents of the extracted H A are generally higher, reflecting the high amount of non-humified biomolecules (polysaccharides and polypeptides) present in the added organic wastes (Petronio et al., 1989). Both the original manure and sludge material used in the experiment had a C/N ratio higher than the one found in H A extracted from the treated soils, indicating that the soil microbial processes have fostered the degree of maturity of the organic wastes during the period after addition (Zucconi & de Bertoldi, 1987). However, the significant difference shown in the C/N, C/H and C/O ratios between manure and sludge (Table 3) suggests that the latter contains a more mature,
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A. Piccolo, P. Zaccheo, P. G. Genevini
Table 3. Elemental composition of extracted HA (%) and of 'model' humic (mHA) and fulvic (mFA) acids on dry and ash-free basis
Soil SM1 SS1 SS2 SS3 Mh S mHA mFA
C
H
47.24 a b" 48.63 b 45.38 a 47-99ab 48-80 b 50-25 a 58.08 b 56.21 45-70
4-97 a 5.18 b 5"33 c 5"61 d 5-80 e 5-90 a 8"35 b 4'76 5"40
N
5.38 a 4"95 a 5.20bc 5.55bc 5'67 c 3.58 a 5"90 b 3.25 2-10
S
0
C/N
C/H
O/C
C/O
0.97 a 0"97 a 0.97 a 1'12 b 1.14 b 0"73 a 1.14 b 0"84 1"90
40.44 a 39"27 b 42.12 c 38"73 d 37.59 e 38-54 a 25"03 b 35'5 44.8
8.78 9"82 8-72 8.64 8"60 14-03 9"96 17.29 21.76
9-50 9"43 8.54 8.62 8-47 8-54 6.94 11-90 8"5
0"85 0"80 0-92 0"80 0-77 0-76 0.43 0"63 0"98
1.17 1.25 1.08 1.25 1'29 1.31 2.32 1.59 1.02
"Means in the same column followed by the same letter are not significantly different (P= 0.05, n = 8). hM, manure; S, sludge. h u m i f i e d o r g a n i c m a t t e r . T h i s d i f f e r e n c e in maturity between the two materials continues to be observed in the C / N ratios of H A extracted from the treated soils, although the C / N ratio for the s l u d g e - a m e n d e d soils d e c r e a s e d despite the increasing sludge doses. F u r t h e r m o r e , the content of C in H A from the m a n u r e - t r e a t e d soil was higher than the control whereas the same value was reached for the sludge t r e a t m e n t only w h e n four times as m u c h sludge was added. This suggests that microbial activity m a y be higher in the sludge-treated soil than in m a n u r e d soil, since part of the carbon added with the sludge could have been lost through microbial respiration. T h e values for functional groups, methoxyls, and E 4 / E 6 ratios of extracted H A and those for 'model' H A and FA are r e p o r t e d in Table 4. Functional groups content did not resemble 'model' F A and in most cases they differed also f r o m 'model' HA. Total, carboxylic and phenolic acidities in H A of treated soils s h o w e d lower values than even the 'model' H A . O n l y the values obtained for H A f r o m the control soil w e r e similar to the 'model' H A . H A f r o m the original m a n u r e and sludge material had even lower values for the three acidities (Table 4) and the sludge H A values even fall below the lower range of soil H A (5.6-8"9, 1.5-5.7, and 2-1-5.7 mol kg-~, respectively; Schnitzer, 1978). T h e s e low results f o u n d for the sludge H A reflect the a n a e r o b i c conditions in which the sludge was p r o d u c e d (Boyd et al., 1980). T h e low levels of the oxygen-containing acidic groups in the extracted H A are similar to those r e p o r t e d f r o m d e c o m p o s i n g plant residues (Kononova & A l e k s a n d r o v a , 1973; I n b a r et al., 1990) and various organic wastes (Riffaldi et al., 1983). T h e total, carboxylic and phenolic acidities of H A f r o m soil samples treated with increasing doses of sludge generally r e m a i n l o w e r than the
Table 4. Content of acidic functional groups (meq/g HA)
Soil SM1 SS1 SS2 SS3 M~ S mHA mFA
Total acidity
COOH
Phenolic OH
6"27"b 5"89 ab 5-36 a 5.33 a 5.16 a 5"30b 3"33 a 6'70 10'30
2"89 d 2.71 c 2.49 b 2.40 b 2.20 a 1.73b 1.35 a 3"60 8"20
3"38 a 3"18 a 2"87 a 2"93 a 2"96 a 3"57b 1"98 a 3"90 3"00
"Means in the same column followed by the same letter are not significantly different (P = 0"05, n = 8). ~'SeeTable 3.
values found for the m a n u r e - t r e a t e d soil and show s o m e decreasing trend. T h e c o n t e n t distribution of the ketonic and methoxylic groups was similar to that of the acidic group (Table 5). T h e sludge-treated samples showed a d e c r e a s e o f ketones with increasing sludge doses, although the values r e m a i n e d significantly higher t h a n that f o u n d in the m a n u r e treated soil. This suggests that, despite the high ketonic c o n t e n t of the m a n u r e material, these groups are m o r e efficiently oxidized in soils than the ketones p r e s e n t in the m o r e stable and humifled sludge. T h e m e t h o x y l g r o u p content, that bears an inverse relationship with H A maturity (Flaig et al., 1975; H a y e s & Swift, 1978), is m u c h lower in the sludge than in the m a n u r e material, confirming the previous observations on the different degrees of humification of the two organic wastes. This m a y be ascribed to the larger methoxyl-rich lignin in the partially u n d e c o m posed c r o p residues which are present in considerable a m o u n t s in m a n u r e . T h e H A f r o m the treated soils s h o w e d a m e t h o x y l c o n t e n t that
279
Humic substances from waste-amended soils Table 5. Content of ketonic and methoxylic groups and E4/E~
Soil SM1 SS1 SS2 SS3 Mh S mHA mFA
Ketones (meq/g)
Methoxyls %
E4/E~,
4-28 a" 4-04 a 5.71 b 4.86 b 4.63 a 2.84 b 1.72 a 2.90 2.70
2.43 b 2"92 b 1-65 a 1.84 a 1.81 a 6'07 b 2.65 a 6.07 0"80
5"80 c 6"08 c 6.11 c 5.40 b 4.96 a 4.66 b 3-04 a 4.96 9-60
"Means in the same column followed by the same letter are not significantlydifferent (P= 0"05, ii =-8). bM, manure; S, sludge. reflected the difference existing between the two original materials being significantly lower for the sludge-amended soils than for the manure-treated samples. The E4/E6 ratio, a parameter inversely related to the molecular dimension of H A (Chen et al., 1977), revealed a larger molecular size for the sludge material (3.04) as compared to the manure (4.66). This agrees with the noted largely humifled character of the sludge waste that presumes a high molecular dimension. This characteristic influences the E4/E(, ratio of H A extracted from the treated soils. Those for the sludge-treated soils showed increasingly lower ratios suggesting a progressive HA molecular-size enhancement with amount of sludge addition. The results in Table 3 and 4 indicate that the H A extracted from the organic wastes-amended soils, although showing an elemental composition more typical of a 'model' soil FA, revealed a content of acidic functional groups that more closely resembled a soil 'model' HA. In particular, the degree of maturity of the added wastes determined the characteristics of the H A extracted from the amended soils indicating that the sludgederived H A were generally m o r e humified than H A from the manure-amended soils. M o l e c u l a r weight distribution
The different molecular dimensions suggested by the E4/E 6 ratios for the manure and sludge materials were also evident in the molecular weight distribution of the humic extracts obtained by gel permeation chromatography on Sephacryl $200 (Fig. 1). H A from sludge showed only one peak (Fig. 1 (A))that corresponded approximately to the void elution volume (V0), and represented the eluted fraction with a nominal molecular
weight higher than 250 000. Conversely, H A from manure (Fig. I(B)) showed a second peak representing an eluting fraction that was able to diffuse through the gel and had, thus, a nominal weight lower than 250000. These chromatograms clearly confirm the E 4 / E 6 results that manure contained humffied fractions of lower molecular weight than sludge. The molecular dimension of humus added to soils with organic wastes influences the molecular size of humic acids in soils (Piccolo & Mbagwu, 1990). Following the treatment with manure (25 t ha -~ year-~), humic extracts showed a slight enhancement of the high molecular weight fraction (Fig. 2(B)) as compared to the chromatogram of the H A of control soil (Fig. 2(A)). This tendency was also shown and even increased in the H A from the sludge-treated soils (Fig. 2(C,D,E,)). The component of high molecular weight was enhanced with progressively increasing amounts of added sludge, indicating that sludge additions to soils are capable of increasing the molecular size of soil humus. Moreover, a general reduction in absorbance in both chromatographic peaks is observed in the chromatograms of extracts from sludge-added soils as compared to humus from the control soil (Fig. 2(A)). This reflects the low original absorbance shown by the extract from the sludge material (Fig. 1). Piotrowsky et al. (1984), by means of ~3C N M R spectroscopy, determined that sludges deriving from urban and industrial wastes were predominantly rich in carbohydrate
E
Z
el,,,
J,t
,
,
40 Vo 80 120 [LUTION VOLUME, ml Fig. 1. Gel permeation chromatograms of HA from the (A)
original sludge and (B) original manure material.
280
A. Piccolo, P. Zaccheo, P. G. Genevini
~'~A
am
Ig
C~ ,,,a ,at
"
E J
4OV0
80 120 ELUTION VOLUME,ml
Fig. 2. Gel permeation chromatogramsof HA from the (A) soil, (B) soil plus manure (25 t ha-i year-~), (C) soil plus sludge (25 t ha -j year-J), (D) soil plus sludge (50 t ha -j year- ~),(E) soil plus sludge ( 100 t ha- i year- ~). and aliphatic components. Preston et al. (1987) followed, by J3C NMR spectroscopy, the carbon changes in soil organic matter in a long-term field experiment and found that the carbohydrate carbons were lost whereas the lipid and methoxyl carbons selectively increased. These findings may explain the differences observed in peak absorbance between the H A from sludge-treated soils and that of the control soil. The sludge material, consisting mainly of low light-absorbing compounds such as carbohydrates and aliphatics (Petronio et al., 1989), is transformed in soil to humic substances that give chromatographic peaks of lower absorbance than those of H A of the mature control soil that has been progressively enriched with higher light-absorbing components.
Infraredspectra Infrared spectra of HA extracted from the original manure and sludge materials, the control soil, and the amended soil samples are shown in Fig. 3. The main absorption bands are: a broad band at 3 3 0 0 - 3 4 0 0 cm -~ (H-bonded O H and N H groups), a slight shoulder at 3085 c m - l (aromatic C - - H stretching), a sharp peak at 2920 cm-~ and a shoulder at 2850 cm-~ (aliphatic C - - H stretching), a shoulder in the 2 6 0 0 - 2 5 0 0 cm-~ region
~
241N lira F|IIIE|CI
14N Ici q
lira
UO
Fig. 3. Infrared spectra of HA from the (A) original sludge and (B) original manure material;(C) soil, (D) soil plus manure (25 t ha -I year-I), (E) soil plus sludge (25 t ha -~ year-J), (F) soil plus sludge (50 t ha-~ year-~), (G) soil plus sludge ( 100 t ha-n year- J).
(H-bonded OH of C O O H groups), a shoulder or peak at 1710 cm -~ ( C = O of COOH, C = O of ketonic carbonyls), a well pronounced broad peak at around 1650 c m - i (aromatic C = C , H-bonded C = O , NH 2 deformation), a peak at 1 5 1 0 - 1 5 4 0 c m - i (NH2 deformation of amides), a peak at 1460 cm -t (aliphatic C - - H deformation), a 1420-1300 cm -~ region (C--H deformation of aliphatic groups), a broad band centered at 1240 c m - l (--C--O--, - - C - - N stretchings and O - - H deformations), peaks at the 1 1 6 0 - 1 1 1 0 cm -~ region (OH or C - - O stretchings of various groups, aromatic ring bending), and peaks around 1100-1020 cm -~ (C--O stretchings of polysaccharides). Interpretations of the IR spectra are based on Bellamy (1975), Stevenson (1982), and Durig etaL (1988).
Humic substances from waste-amended soils
T h e infrared s p e c t r u m of H A extracted from the sludge material (Fig. 3(A))is similar to spectra of H A f r o m other sludges (Gerasimowicz & Byler, 1985; Petronio et al., 1989) and shows a particularly high content of aliphatic groups ( 2 9 8 0 - 2 9 6 0 c m - ~ region and 1460 c m - l) and of N-containing c o m p o u n d s such as peptides (sharp b a n d at 1650 cm -~ c o n c o m i t a n t to the 1550 cm -1 band). T h e m a n u r e H A s p e c t r u m (Fig. 3(B)) reveals a lower content of aliphatic and N-containing groups but m o r e p r o n o u n c e d bands attributable to polysaccharides (large b a n d in the 1 2 1 0 - 1 2 7 0 c m region and a sharp absorption at 1040 c m - ~). H A extracted from the control soil p r o d u c e d an IR spectrum (Fig. 3(C)) similar to those r e p o r t e d in the literature (Schnitzer, 1978) and particularly to the type III H A of Stevenson & G o b ( 1971 ). Following m a n u r e addition to soil samples, the resulting H A had an IR s p e c t r u m (Fig. 3(D)) which did not a p p e a r different f r o m that of the control soil except for a slight increase of s o m e absorptions due to aliphatic groups in the 1 4 6 0 - 1 4 1 0 cm -~ region and of a peak at 1130 cm-~ attributable to O H and C O stretchings of carbohydrates. T h e IR spectra of H A extracted from soils a m e n d e d with increasing sludge doses (Fig. 3(E,F,G)) showed a m a r k e d e n h a n c e m e n t of the aliphatic groups ( 2 9 6 0 - 2 8 5 0 c m -~ region) and of the band of peptides (1540 cm - j ) concomitant with the a m o u n t of applied sludge. Moreover, the large absorption at a r o u n d 3400 c m - i is progressively shifted towards lower frequencies (3300 c m - I ) , p r o b a b l y due to the increasing contribution of the N - - H stretching modes of peptides a d d e d with the sludge over the O - - H stretching of the oxygen containing functional groups. As in the case of m a n u r e , sludge additions to soil result in the levelling of the 1 2 0 0 - 1 1 0 0 cm -~ region as c o m p a r e d to the original waste material as r e p o r t e d by o t h e r workers ( K o n o n o v a & A l e k s a n d r o v a , 1973; Sugahara & Inoko, 1981; Inbar et al., 1990). T h e reduction in intensity of the peaks of the polysaccharides region was r e p o r t e d also for d e c o m position of peat and was attributed to the b r e a k d o w n of ligno-cellulose (Durig et al., 1988).
REFERENCES American Society of Agronomy (1982). Methods of Soil Analysis. ASA, Madison, Wl. Bellamy, L. J. ( 1975). The Infrared Spectra of Complex Molecules. Chapman & Hall, London. Boyd, S. A., Sommers, L. E. & Nelson, D. W. (1980).
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Changes in the humic acid fraction of soil resulting from sludge application. Soil. Sci. Soc. Amer. J., 44, 1179-86. Campanella, L., Ferri, T., Petronio, B. M. & Piccolo, A. (1989). Effect of speciation in sludges on the absorption of leached metals from soil. Sci. Total Environ., 79, 223-31. Chen, Y., Senesi, N. & Schnitzel M. (1977). Information provided on humic substances by E4/E6 ratios. Soil Sci. Soc. Amer. J., 41,352-7. Durig, D. T., Esterle, J. S., Dickson, T. J. & Durig, J. R. (1988). An investigation of the chemical variability of woody peak by FT-IR spectroscopy. Appl. Spectrosc., 42, 1239-44. Flaig, W., Beutelspacher, H. & Rietz, E. (1975). Chemical composition and physical properties of humic substances. In Soil Components, Vol. 1, ed. J. E. Gieseking. Springer Verlag, Berlin, pp. 1-212. Fritz, J. S., Yamamura, S. S. & Bradford, E. C. (1959). Determination of carbonyl compounds. Anal. Chem., 31, 260-3. Gerasimowicz, W. V. and Byler, D. M. (1985). Carbon-13 CPMAS NMR and FT-IR spectroscopic studies of humic acids. SoiISci., 139, 270-6. Hayes, M. H. B. & Swift, R. S. (1978). The chemistry of soil organic colloids. In The Chemistry of SoU Constituents, ed. M. H. B. Hayes. John Wiley, New York. lnbar, Y., Hadar, Y. &Chen, Y. (1990). Characterization of humic substances formed during the composting of solid wastes from wineries. Sci. Total Environ. (in press). Kononova, M. M. & Aleksandrova, I. V. (1973). Formation of humic acids during plant residue humification and their nature. Geoderma, 9, 157-64. Marchesini, A., Allievi, L., Comotti, E. & Ferrari, A. (1988). Long-term effect of quality-compost treatment on soil. Plant &SoU, 106, 253-61. Mbagwu, J. S. C. & Piccolo, A. (1989). Changes in soil aggregate stability induced by amendments with humic substances. Soil Technol., 2, 49-57. Mbagwu, J. S. C. & Piccolo, A. (1990). Carbon, nitrogen, and phosphorus concentrations in aggregates of organic wasteamended soils. Biol. Wastes, 31, 97-111. Petronio, B. M., Ferri, T., Papalini, C. & Piccolo, A. (1989). Characterization of the organic component of a sludge. Talanta, 36(12), 1177-82. Petrussi, E, De Nobili, M., Viotto, M. & Sequi, E (1988). Characterization of organic matter from animal manures after digestion by earthworms. Plant & Soil, 105, 41-6. Piccolo, A. (1984). Caratterizzazione chimica e chimicofisica di un acido umico da torba. Uno studio di purifieazione. Ann. Inst. Stud. Dif. Suolo, XXV, 121-31. Piccolo, A. & Stevenson, E J. (1982). Infrared spectra of Cu 2+, Pb 2+, and Ca 2÷ complexes of soil humic substances. Geoderma, 27, 195-208. Piccolo, A. & Mirabella, A. (1987). Characteristics of soil humic extracts obtained by some organic and inorganic solvents and purified by the HCI-HF treatment. Soil Sci., 146,418-26. Piccolo, A. & Mbagwu, J. S. C. (1990). Effects of different organic waste amendments on soil microaggregates stability and molecular sizes of humic substances. Plant & Soil 123,27-37. Piotrowsky, E. G., Valentine, K. M. & Pfeffer, P. E. (1984). Solid-state, C-13, cross-polarization, 'magic angle' spinning, NMR spectroscopy studies of sewage sludge. Soil Sci., 137, 194-203. Preston, C. M., Shipitalo, S. E., Dudley, R. L., Fyfe, C. A., Mathur, S. P. & Levesque, M. (1987). Comparison of C-13 CPMAS NMR and chemical techniques for measuring the degree of decomposition in virgin and cultivated peat profiles. Can. J. Soil Sci,, 67, 187-98.
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Riffaldi, R., Levi-Minzi, R. & Saviozzi, A. (1983). Humic fractions of organic wastes. Agric., Ecosyst. Environ., 10, 353-9. Schnitzer, M. (1978). Humic substances: chemistry and reactions. In Soil Organic Matter, ed. S. U. Khan & M. Schnitzer, Elsevier, Amsterdam. Senesi, N. & Sposito, G. (1984). Residual copper (ll) complexes in soil and sewage sludge fulvic acids: an electron spin resonance study. SoilSci. Soc. Amer. J., 48, 1247-53. Stevenson, F. J. (1982). Humus Chemistry. John Wiley, New York. Stevenson, E J. & Goh, K. M. (1971). Infrared spectra of humic acids and related substances. Geochim. Cosmochim. Acta, 35,471-83.
Sugahara, K. & lnoko, A. (1981). Composition analysis of humus and characterization of humic acid obtained from city refuse compost. Soil ScL &Plant Nutr., 27, 213-24. Swift, R. S. & Posner, A. M. (1971 ). Gel chromatography of humic acids. J. SoilSci., 22,237-49. Wright, J. R. & Schnitzer, M. (1959). Oxygen-containing functional groups in the organic matter of a podzol soil. Nature (London), 184, 1462-3. Zucconi, F. & de Bertoldi, M. (1987). Compost specifications for the production and characterization of compost from municipal solid waste. In Compost: Production, Quality, and Use, ed. M. de Bertoldi, ~M. P. Ferranti, P. L'Hermite & E Zucconi. Elseiver Applied Science, London, pp. 359-67.
Chemical Characterization of Humic Substances Extracted from Organic-Waste-Amended Softs A. Piccolo Istituto Sperimentale per 1o Studio e la Difesa del Suolo, Piazza M. D'Azeglio 30, Firenze, Italy
E Zaccheo & P. G. Genevini Istituto Chimica Agraria, Universitfi di Milano, Via della Celoria, Milano, Italy (Received 24 October 1990; accepted 18 June 1991)
Abstract
Humic substances were extracted from a soil treated, in a 4-year experiment, at different rates with a sludge from anaerobic treatment of combined civil and industrial wastes, and with agricultural manure. Elemental and chemical analyses, molecular weight (MW) distribution and infrared (IR) spectroscopy were performed on the purified humic acids (HA). Organic wastes significantly increased the HA content of the treated soils and improved CEC. The C/N, C/H and C/O ratios of HA extracted from the original wastes showed a higher degree of humification for sludge than for manure. This difference was also noticed for the C/N ratio of soil humic extracts, indicating a faster humification process for the sludges in soil. The content of oxygen-containing functional groups was lower than the 'model' HA reported in the literature, and even more so for HA from sludges, reflecting their anaerobic formation. M W distribution and E4/E 6 ratios showed that the sludge material had a higher molecular complexity than manure, supporting the high degree of humification attributed to the former. HA extracted from sludgetreated soils revealed a molecular dimension increasing with the application doses of waste material. Infrared spectra showed that HA formed in soils after waste additions reflected the chemical composition of the original organic material, which was rich in aliphatic groups and peptides for sludge and in carbohydrates for manure. On the basis of this study, it is concluded that not only are organic waste additions able to build up the HA content in soils but the HA formed assume the chemical characteristics and the degree of humification of the original material.
Key words: Humus, soil, organic waste.
INTRODUCTION The worldwide growing demand for means of disposing organic wastes derived from agricultural productions and urban and industrial refuses has increasingly turned to soils as a highly suitable acceptor. It is claimed that the soil chemical and biological activity may efficiently and safely incorporate organic wastes into the soil organic components. This practice often results in an improvement in the chemical and physical properties of the soil (Marchesini et al., 1988; Mbagwu & Piccolo, 1989, 1990), although there is an increasing awareness of the risk of groundwater contamination by pollutants such as heavy metals leached down in soluble organic matter complexes (Campanella et al., 1989). The realization that organic matter introduced into soils by waste additions may alter the soil capacity to prevent pollutants from a downward mobility has led to increased research on organic matter characterization, since information on humus chemical properties may be useful to predict the fate of a pollutant in the soil environment. Sugahara & Inoko (1981) reported on humic acid properties of a city refuse compost, Petronio et al. (1989) proposed a fractionation scheme of the organic components of urban and industrial sludges for a better evaluation of the chemical properties, Petrussi et al. (1988) investigated the characteristics of organic matter from animal manures after digestion by earthworms, Inbar et aL (1990) followed the chemical changes of humus during the compost-
275 Bioresource Technology 0960-8524/92/S05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain
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ing of solid wastes from wineries. Other works studied the strength of metal complexes with soluble fulvic acids extracted from anaerobically digested sewage sludges (Senesi & Sposito, 1984). Less work has been done on the chemical characteristics of soil humic substances extracted after addition of organic wastes to soils. Piccolo & Mbagwu (1990) reported that addition of pig and cattle slurry and sewage sludge to soils in a long term experiment resulted in a selective increase of the high-molecular-weight humic fraction in soil micro-aggregates. The objective of this work was to investigate the general chemical and physical-chemical characteristics of humic substances extracted from soils treated with anaerobically digested sludge and cattle manure.
METHODS Soil and organic wastes A sandy loam soil classified as Fluventic Xerochrept was sampled in an agricultural area south of Milano, Italy. The anaerobic sludge (S) came from a civil and industrial waste water treatment plant, whereas cattle manure (M) originated from a livestock breeding farm. Some of the chemical characteristics of the soil, sludge and manure are reported in Table 1. Analyses were performed according to ASA Methods of Soil Analysis (1982). The sampled soil was used to fill greenhouse pots set for a 4-year experiment with the following amendments and rates (dry matter): control pot with only mineral fertilization (soil); 25 t hayear-t of sludge (SS1); 50 t ha-1 year- ~of sludge (SS2); 100 t ha-~ year-~ of sludge (SS3); 25 t ha-J year-J of cattle manure (SM1). Four replicates were arranged for each treatment. During the experimental period, Lolium italieum, for a total amount of 16 cuts, was grown in each pot. At the end of the experiment, a soil sample (0-15 cm)for each replicate of each treatment was collected. These were thoroughly mixed and four subsamples for each treatment were obtained, air dried, sieved (2 mm) and stored in the freezer for subsequent analysis. Extraction of humic acids Soil samples were suspended in 0.05 M HC1 for 24 h, filtered, washed with deionized H20 until chloride-free and then air dried. The samples were ground and suspended in 0.5 M NaOH
(soil:solution, 1:5) under a n N 2 atmosphere for 24 h. The alkaline extract was then centrifuged at 3000 g for 30 min, the precipitate was acidified with HC1 to pH 2, decanted at room temperature for 24 h and centrifuged to separate humic from fulvic acids (FA). The humic acids (HA) were then redissolved in 0.1 N NaOH under N2 and reprecipitated with 0-5 N HC1. The HA were further purified by a 24-h treatment with a 0.5% HCI/HF mixture with stirring. This treatment was repeated twice to lower the humic acid ash content. Purified HA were transferred to Visking dialysis tubes with an exclusion limit of 8000-15000 Dalton, and dialysed against deionized water for 5 days, and then freeze-dried. The extraction yield averaged approximately 4 g kg- ~of soil. Chemical characterization The C, H, N, S, elemental composition of HA was determined on A N A 1500 Carlo Erba instrumentation. Total acidity was determined by the Ba(OH)2 method, and values for carboxylic groups were obtained through the Ca-acetate procedure (Wright & Schnitzer, 1959). Phenolic groups were calculated by subtracting carboxylic groups from total acidity. Ketonic groups were determined by the hydroxylamine hydrochloride method (Fritz et al., 1959) and methoxyls by the Zeisel method (Piccolo, 1984); E 4 / E 6 w a s the ratio of absorbances at 465 nm and 665 nm (Chen etal., 1977). Chemical characterization was repeated twice for each sub-sample, reaching a total of 8 data/ treatment. The data obtained were statistically computed with ANOVA and Tuckey's alternate test.
Table 1. Analytical parameters for soil, sludge (S), and manure (M)
pH OM (%) N Tot. (%) P Tot. (%) CEC (meq/100) HA (%) AI (ppm) Cd (ppm) Cu (ppm) Cr (ppm) Ni (ppm) Pb (ppm) Zn (ppm)
Soil
S
M
7.5 2-06 0.13 0.12 16.7 0.32 Tr. Tr. Tr. Tr. Tr. Tr. Tr.
7.1 36,2 1,4 3,8 ND 1-32 2.01 47.2 724 1489 494 586 3410
9.06 72.4 2.67 1.4 ND 12-7 Tr. Tr. Tr. Tr. Tr. Tr. Tr.
ND, not determined;Tr., traces.
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Humic substances from waste-amended soils
Molecular weight distribution Molecular weight distribution of HA was determined using Sephacryl $200 as a molecular sieve in the gel permeation chromatography technique. This matrix, which was packed in a 70 cm x 1.5 cm Pharmacia column, has an upper exclusion limit of 2 5 0 0 0 0 daltons and, being a Dextran further crosslinked with N,N'-methylene bisacrylamide, Sephacryl has a higher rigidity than Sephadex, and allows higher flow rates and better control of pore size. The gel packing solution and the eluent was a 1 M 2-amino-2-hydroxymethyl1,3-propanediol (TRIS) buffered at pH 9 (Swift & Posner, 1971 ). Elution pressure was assured by a peristaltic pump giving a flow rate of 6-8 mi h Samples for chromatography (20 mg) diluted in 1 ml of TRIS were inserted on to the column surface by a four-way injection valve (Pharmacia SRV 4). The absorbance of the eluate was measured at 470 nm and the subsequent chromatograms were automatically produced by a continuous flow detector ISCO UA-5 with an incorporated recorder. The void volume of the column (V0) was assessed with Blue Dextran 2000. Other details on the column and gel calibration are reported elsewhere (Piccolo & Mirabella, 1987). Infrared spectra Infrared spectra were recorded using a Perkin Elmer 882 double-beam spectrometer. Samples for infrared analysis were prepared by mixing 1 mg of freeze-dried humic material with 300 mg of pre-dried and pulverized spectroscopic grade KBr. The KBr pellets were prepared as outlined by Piccolo & Stevenson (1982).
RESULTS AND DISCUSSION Extractable HA and soil CEC Yields of H A and the CEC for each soil at the end of the experiment are reported in Table 2. T h e percentage of extractable H A in the control soil decreased noticeably after 4 years, whereas soil samples treated with both manure and different amounts of sludge showed increased content of HA. Addition of sludges produced a H A content enhancement parallel to the application doses, which reached 200% of control for the highest sludge addition. Manure treatment increased soil H A content to a level not significantly different from the one corresponding to the equivalent amount of sludges. Also the CEC seemed to be
Table 2. Percentageof soil HA and soil CEC
Soil SM1 SS1 SS2 SS3
HA (%)
CEC ¢rneq/lO0)
0.21 a" 0'38ab 0-36 ab 0.42 b 0-66 c
18-8 a 21.2 a 19.7 a 21.6 a 25.0 b
"Means in the same column followed by the same letter are not significantlydifferent (P = 0"05, n = 8).
positively affected by the sludge and manure treatments, although its increase was significantly different from the control only at the highest level of sludge addition. These results indicate that organic wastes effectively build up the H A content of soils and that they have a beneficial effect on a soil-fertility parameter such as CEC. Elemental composition and functional group analysis of HA H A extracted from soils commonly have similar elemental compositions regardless of climatic and soil conditions (Schnitzer, 1978). The elemental composition and atomic ratios of H A extracted from the treated soils as well as that of 'model' H A and FA are presented in Table 3. Notwithstanding the type of organic waste and applied doses all the parameters found were strikingly similar. It is noteworthy, however, that the average composition of the HA extracted from the treated soils resembles more closely that of the 'model' FA than that of the 'model' HA. Both 'model' H A and FA represented a large number of humic materials formed under widely differing conditions (Schnitzer, 1978). Though the C and H contents of H A from the control soil, the soil plus manure, and soil plus different sludge doses, are close to the values reported for the 'model' FA, the N and S contents of the extracted H A are generally higher, reflecting the high amount of non-humified biomolecules (polysaccharides and polypeptides) present in the added organic wastes (Petronio et al., 1989). Both the original manure and sludge material used in the experiment had a C/N ratio higher than the one found in H A extracted from the treated soils, indicating that the soil microbial processes have fostered the degree of maturity of the organic wastes during the period after addition (Zucconi & de Bertoldi, 1987). However, the significant difference shown in the C/N, C/H and C/O ratios between manure and sludge (Table 3) suggests that the latter contains a more mature,
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Table 3. Elemental composition of extracted HA (%) and of 'model' humic (mHA) and fulvic (mFA) acids on dry and ash-free basis
Soil SM1 SS1 SS2 SS3 Mh S mHA mFA
C
H
47.24 a b" 48.63 b 45.38 a 47-99ab 48-80 b 50-25 a 58.08 b 56.21 45-70
4-97 a 5.18 b 5"33 c 5"61 d 5-80 e 5-90 a 8"35 b 4'76 5"40
N
5.38 a 4"95 a 5.20bc 5.55bc 5'67 c 3.58 a 5"90 b 3.25 2-10
S
0
C/N
C/H
O/C
C/O
0.97 a 0"97 a 0.97 a 1'12 b 1.14 b 0"73 a 1.14 b 0"84 1"90
40.44 a 39"27 b 42.12 c 38"73 d 37.59 e 38-54 a 25"03 b 35'5 44.8
8.78 9"82 8-72 8.64 8"60 14-03 9"96 17.29 21.76
9-50 9"43 8.54 8.62 8-47 8-54 6.94 11-90 8"5
0"85 0"80 0-92 0"80 0-77 0-76 0.43 0"63 0"98
1.17 1.25 1.08 1.25 1'29 1.31 2.32 1.59 1.02
"Means in the same column followed by the same letter are not significantly different (P= 0.05, n = 8). hM, manure; S, sludge. h u m i f i e d o r g a n i c m a t t e r . T h i s d i f f e r e n c e in maturity between the two materials continues to be observed in the C / N ratios of H A extracted from the treated soils, although the C / N ratio for the s l u d g e - a m e n d e d soils d e c r e a s e d despite the increasing sludge doses. F u r t h e r m o r e , the content of C in H A from the m a n u r e - t r e a t e d soil was higher than the control whereas the same value was reached for the sludge t r e a t m e n t only w h e n four times as m u c h sludge was added. This suggests that microbial activity m a y be higher in the sludge-treated soil than in m a n u r e d soil, since part of the carbon added with the sludge could have been lost through microbial respiration. T h e values for functional groups, methoxyls, and E 4 / E 6 ratios of extracted H A and those for 'model' H A and FA are r e p o r t e d in Table 4. Functional groups content did not resemble 'model' F A and in most cases they differed also f r o m 'model' HA. Total, carboxylic and phenolic acidities in H A of treated soils s h o w e d lower values than even the 'model' H A . O n l y the values obtained for H A f r o m the control soil w e r e similar to the 'model' H A . H A f r o m the original m a n u r e and sludge material had even lower values for the three acidities (Table 4) and the sludge H A values even fall below the lower range of soil H A (5.6-8"9, 1.5-5.7, and 2-1-5.7 mol kg-~, respectively; Schnitzer, 1978). T h e s e low results f o u n d for the sludge H A reflect the a n a e r o b i c conditions in which the sludge was p r o d u c e d (Boyd et al., 1980). T h e low levels of the oxygen-containing acidic groups in the extracted H A are similar to those r e p o r t e d f r o m d e c o m p o s i n g plant residues (Kononova & A l e k s a n d r o v a , 1973; I n b a r et al., 1990) and various organic wastes (Riffaldi et al., 1983). T h e total, carboxylic and phenolic acidities of H A f r o m soil samples treated with increasing doses of sludge generally r e m a i n l o w e r than the
Table 4. Content of acidic functional groups (meq/g HA)
Soil SM1 SS1 SS2 SS3 M~ S mHA mFA
Total acidity
COOH
Phenolic OH
6"27"b 5"89 ab 5-36 a 5.33 a 5.16 a 5"30b 3"33 a 6'70 10'30
2"89 d 2.71 c 2.49 b 2.40 b 2.20 a 1.73b 1.35 a 3"60 8"20
3"38 a 3"18 a 2"87 a 2"93 a 2"96 a 3"57b 1"98 a 3"90 3"00
"Means in the same column followed by the same letter are not significantly different (P = 0"05, n = 8). ~'SeeTable 3.
values found for the m a n u r e - t r e a t e d soil and show s o m e decreasing trend. T h e c o n t e n t distribution of the ketonic and methoxylic groups was similar to that of the acidic group (Table 5). T h e sludge-treated samples showed a d e c r e a s e o f ketones with increasing sludge doses, although the values r e m a i n e d significantly higher t h a n that f o u n d in the m a n u r e treated soil. This suggests that, despite the high ketonic c o n t e n t of the m a n u r e material, these groups are m o r e efficiently oxidized in soils than the ketones p r e s e n t in the m o r e stable and humifled sludge. T h e m e t h o x y l g r o u p content, that bears an inverse relationship with H A maturity (Flaig et al., 1975; H a y e s & Swift, 1978), is m u c h lower in the sludge than in the m a n u r e material, confirming the previous observations on the different degrees of humification of the two organic wastes. This m a y be ascribed to the larger methoxyl-rich lignin in the partially u n d e c o m posed c r o p residues which are present in considerable a m o u n t s in m a n u r e . T h e H A f r o m the treated soils s h o w e d a m e t h o x y l c o n t e n t that
279
Humic substances from waste-amended soils Table 5. Content of ketonic and methoxylic groups and E4/E~
Soil SM1 SS1 SS2 SS3 Mh S mHA mFA
Ketones (meq/g)
Methoxyls %
E4/E~,
4-28 a" 4-04 a 5.71 b 4.86 b 4.63 a 2.84 b 1.72 a 2.90 2.70
2.43 b 2"92 b 1-65 a 1.84 a 1.81 a 6'07 b 2.65 a 6.07 0"80
5"80 c 6"08 c 6.11 c 5.40 b 4.96 a 4.66 b 3-04 a 4.96 9-60
"Means in the same column followed by the same letter are not significantlydifferent (P= 0"05, ii =-8). bM, manure; S, sludge. reflected the difference existing between the two original materials being significantly lower for the sludge-amended soils than for the manure-treated samples. The E4/E6 ratio, a parameter inversely related to the molecular dimension of H A (Chen et al., 1977), revealed a larger molecular size for the sludge material (3.04) as compared to the manure (4.66). This agrees with the noted largely humifled character of the sludge waste that presumes a high molecular dimension. This characteristic influences the E4/E(, ratio of H A extracted from the treated soils. Those for the sludge-treated soils showed increasingly lower ratios suggesting a progressive HA molecular-size enhancement with amount of sludge addition. The results in Table 3 and 4 indicate that the H A extracted from the organic wastes-amended soils, although showing an elemental composition more typical of a 'model' soil FA, revealed a content of acidic functional groups that more closely resembled a soil 'model' HA. In particular, the degree of maturity of the added wastes determined the characteristics of the H A extracted from the amended soils indicating that the sludgederived H A were generally m o r e humified than H A from the manure-amended soils. M o l e c u l a r weight distribution
The different molecular dimensions suggested by the E4/E 6 ratios for the manure and sludge materials were also evident in the molecular weight distribution of the humic extracts obtained by gel permeation chromatography on Sephacryl $200 (Fig. 1). H A from sludge showed only one peak (Fig. 1 (A))that corresponded approximately to the void elution volume (V0), and represented the eluted fraction with a nominal molecular
weight higher than 250 000. Conversely, H A from manure (Fig. I(B)) showed a second peak representing an eluting fraction that was able to diffuse through the gel and had, thus, a nominal weight lower than 250000. These chromatograms clearly confirm the E 4 / E 6 results that manure contained humffied fractions of lower molecular weight than sludge. The molecular dimension of humus added to soils with organic wastes influences the molecular size of humic acids in soils (Piccolo & Mbagwu, 1990). Following the treatment with manure (25 t ha -~ year-~), humic extracts showed a slight enhancement of the high molecular weight fraction (Fig. 2(B)) as compared to the chromatogram of the H A of control soil (Fig. 2(A)). This tendency was also shown and even increased in the H A from the sludge-treated soils (Fig. 2(C,D,E,)). The component of high molecular weight was enhanced with progressively increasing amounts of added sludge, indicating that sludge additions to soils are capable of increasing the molecular size of soil humus. Moreover, a general reduction in absorbance in both chromatographic peaks is observed in the chromatograms of extracts from sludge-added soils as compared to humus from the control soil (Fig. 2(A)). This reflects the low original absorbance shown by the extract from the sludge material (Fig. 1). Piotrowsky et al. (1984), by means of ~3C N M R spectroscopy, determined that sludges deriving from urban and industrial wastes were predominantly rich in carbohydrate
E
Z
el,,,
J,t
,
,
40 Vo 80 120 [LUTION VOLUME, ml Fig. 1. Gel permeation chromatograms of HA from the (A)
original sludge and (B) original manure material.
280
A. Piccolo, P. Zaccheo, P. G. Genevini
~'~A
am
Ig
C~ ,,,a ,at
"
E J
4OV0
80 120 ELUTION VOLUME,ml
Fig. 2. Gel permeation chromatogramsof HA from the (A) soil, (B) soil plus manure (25 t ha-i year-~), (C) soil plus sludge (25 t ha -j year-J), (D) soil plus sludge (50 t ha -j year- ~),(E) soil plus sludge ( 100 t ha- i year- ~). and aliphatic components. Preston et al. (1987) followed, by J3C NMR spectroscopy, the carbon changes in soil organic matter in a long-term field experiment and found that the carbohydrate carbons were lost whereas the lipid and methoxyl carbons selectively increased. These findings may explain the differences observed in peak absorbance between the H A from sludge-treated soils and that of the control soil. The sludge material, consisting mainly of low light-absorbing compounds such as carbohydrates and aliphatics (Petronio et al., 1989), is transformed in soil to humic substances that give chromatographic peaks of lower absorbance than those of H A of the mature control soil that has been progressively enriched with higher light-absorbing components.
Infraredspectra Infrared spectra of HA extracted from the original manure and sludge materials, the control soil, and the amended soil samples are shown in Fig. 3. The main absorption bands are: a broad band at 3 3 0 0 - 3 4 0 0 cm -~ (H-bonded O H and N H groups), a slight shoulder at 3085 c m - l (aromatic C - - H stretching), a sharp peak at 2920 cm-~ and a shoulder at 2850 cm-~ (aliphatic C - - H stretching), a shoulder in the 2 6 0 0 - 2 5 0 0 cm-~ region
~
241N lira F|IIIE|CI
14N Ici q
lira
UO
Fig. 3. Infrared spectra of HA from the (A) original sludge and (B) original manure material;(C) soil, (D) soil plus manure (25 t ha -I year-I), (E) soil plus sludge (25 t ha -~ year-J), (F) soil plus sludge (50 t ha-~ year-~), (G) soil plus sludge ( 100 t ha-n year- J).
(H-bonded OH of C O O H groups), a shoulder or peak at 1710 cm -~ ( C = O of COOH, C = O of ketonic carbonyls), a well pronounced broad peak at around 1650 c m - i (aromatic C = C , H-bonded C = O , NH 2 deformation), a peak at 1 5 1 0 - 1 5 4 0 c m - i (NH2 deformation of amides), a peak at 1460 cm -t (aliphatic C - - H deformation), a 1420-1300 cm -~ region (C--H deformation of aliphatic groups), a broad band centered at 1240 c m - l (--C--O--, - - C - - N stretchings and O - - H deformations), peaks at the 1 1 6 0 - 1 1 1 0 cm -~ region (OH or C - - O stretchings of various groups, aromatic ring bending), and peaks around 1100-1020 cm -~ (C--O stretchings of polysaccharides). Interpretations of the IR spectra are based on Bellamy (1975), Stevenson (1982), and Durig etaL (1988).
Humic substances from waste-amended soils
T h e infrared s p e c t r u m of H A extracted from the sludge material (Fig. 3(A))is similar to spectra of H A f r o m other sludges (Gerasimowicz & Byler, 1985; Petronio et al., 1989) and shows a particularly high content of aliphatic groups ( 2 9 8 0 - 2 9 6 0 c m - ~ region and 1460 c m - l) and of N-containing c o m p o u n d s such as peptides (sharp b a n d at 1650 cm -~ c o n c o m i t a n t to the 1550 cm -1 band). T h e m a n u r e H A s p e c t r u m (Fig. 3(B)) reveals a lower content of aliphatic and N-containing groups but m o r e p r o n o u n c e d bands attributable to polysaccharides (large b a n d in the 1 2 1 0 - 1 2 7 0 c m region and a sharp absorption at 1040 c m - ~). H A extracted from the control soil p r o d u c e d an IR spectrum (Fig. 3(C)) similar to those r e p o r t e d in the literature (Schnitzer, 1978) and particularly to the type III H A of Stevenson & G o b ( 1971 ). Following m a n u r e addition to soil samples, the resulting H A had an IR s p e c t r u m (Fig. 3(D)) which did not a p p e a r different f r o m that of the control soil except for a slight increase of s o m e absorptions due to aliphatic groups in the 1 4 6 0 - 1 4 1 0 cm -~ region and of a peak at 1130 cm-~ attributable to O H and C O stretchings of carbohydrates. T h e IR spectra of H A extracted from soils a m e n d e d with increasing sludge doses (Fig. 3(E,F,G)) showed a m a r k e d e n h a n c e m e n t of the aliphatic groups ( 2 9 6 0 - 2 8 5 0 c m -~ region) and of the band of peptides (1540 cm - j ) concomitant with the a m o u n t of applied sludge. Moreover, the large absorption at a r o u n d 3400 c m - i is progressively shifted towards lower frequencies (3300 c m - I ) , p r o b a b l y due to the increasing contribution of the N - - H stretching modes of peptides a d d e d with the sludge over the O - - H stretching of the oxygen containing functional groups. As in the case of m a n u r e , sludge additions to soil result in the levelling of the 1 2 0 0 - 1 1 0 0 cm -~ region as c o m p a r e d to the original waste material as r e p o r t e d by o t h e r workers ( K o n o n o v a & A l e k s a n d r o v a , 1973; Sugahara & Inoko, 1981; Inbar et al., 1990). T h e reduction in intensity of the peaks of the polysaccharides region was r e p o r t e d also for d e c o m position of peat and was attributed to the b r e a k d o w n of ligno-cellulose (Durig et al., 1988).
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