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Plant and Soil 123, 27-37 (1990). © Kluwer 3[cademic Publishers. Printed in the Netherlands. PLSO 8194

Effects of different organic waste amendments on soil microaggregates stability and molecular sizes of humic substances

A. PICCOLO and J.S.C. MBAGWU Istituto Sperimentale per lo Studio e la Difesa del Suolo, Piazza M. D'Azeglio 30, 1-50121 Firenze, Italy and Department of Soil Science, University of Nigeria, Nsukka, Nigeria

Received 3 April 1989. Revised January 1990

Key words: aggregate stability, amendments, cattle slurry, humic substances, management practices, microaggregates, molecular sizes, pig slurry, sewage sludge, soils

Abstract

Three soils which had been amended for several years with pig slurry, cattle slurry, and sewage sludge were dry-sieved to obtain microaggregates in the size range of 250-125, 125-50, and <50/xm. With amendments, aggregate size distribution of whole soils was shifted to larger sizes, especially for the most fragile soil, whereas percent content of microaggregates decreased except for the lower size aggregates of the fragile soil. Particle size distribution of microaggregates revealed an increase in percent sand and a reduction of percent silt and clay in the <50/zg size fraction for all soils. These results showed the aggregation effect induced by the organic waste additions. Aggregate stability of microaggregates revealed significant correlation with humic substances content (humic acids alone and humic plus fulvic acids) and non significant with total organic matter substantiating the belief that humic substances are the predominant binding agents in this aggregation range. Molecular weight distribution of humic acids extracted from microaggregates of unamended soils demonstrated that the lower the soil aggregate size distribution, the larger the contribution of the high molecular weight fraction. All microaggregates from amended soils showed a progressive increase of the high molecular weight humic acids with decreasing size, reaching a maximum in the <50/zm fraction. In this aggregate size a parallel enhancement of the aggregate stability was also evident. It is concluded that a close relationship exists between aggregate stability and high molecular weight humic substances. Additions to soils of organic material containing high molecular weight constituents would represent a useful management practice to improve aggregate stability.

Introduction

Among the soil physical properties which organic matter is reported to influence, the degree of aggregation has been extensively studied. Con- siderable controversy, however, exists on the actual role of organic matter in soil aggregation and aggregate stability. Some workers (Chaney and Swift, 1984; Christensen, 1986) found a direct correlation between soil organic matter contents and aggregate stability, others (Dormaar, 1983; Hamblin and Greenland, 1977; Hamblin

27

and Davies, 1977) observed that it is the fractions of organic matter, rather than the total amount per se, that are more important in mod- ifying the structural stability of aggregates.

There are also differences of opinion regarding the actual organic matter constituents that are responsible for improving soil aggregation. Acton et al. (1963) and Mehta et al. (1960) noted that soil polysaccharides correlated positively with aggregate stability. Dormaar (1983) also associated polyuronides and phenols with the >250 ~m size fraction of water-stable aggre-

28 Piccolo and Mbagwu

gates, whereas Hamblin and Davies (1977) de- nied that polysaccharides are essential in aggregate stabilization.

More recently, Tisdall and Oades (1982) reported that polysaccharides act as transient binding agents which are rapidly decomposed by microorganisms and are predominantly associated with the >250 /zm transient stable aggregates. It has been suggested that humic materials associated with amorphous iron, aluminum and aluminosilicates are the persistent binding agents of microaggregates (<250 ~m) (Chaney and Swift, 1986b; Edwards and Brem- ner, 1967; Tisdall and Oades, 1982). The organo- mineral fraction probably includes complexes of clay-polyvalent metal-organic matter (C-P-OM), (Theng, 1979) but the chemical nature of those persistent binding agents have not yet been clearly defined.

Characterization of humic substances reported in literature is limited to higher size aggregates and to aggregates lower than 250/~m as a whole. Owing to the wet sieving technique used to separate aggregates, no further evaluation of the stability of the separated aggregates was ever attempted. Dell'Agnola and Ferrari (1971) found that the more stable macroaggregates related well with the higher molecular weight humic compounds and Chakraborty et al. (1979, 1982) observed that humic acids of low molecular weight were mainly responsible for stabilizing large size aggregates. No studies are yet reported in the literature to correlate the molecular sizes of humic substances with soil microaggregation although it is in this aggregates group that humic substances are believed to exert their binding role (Tisdall and Oades, 1982).

The work presented here relates the molecular dimension of humic substances to the stabilities of their associated <250 ~m aggregates. Further- more, since the agricultural soils selected to obtain the microaggregates had received amendments of different organic materials such as pig and cattle slurry and sewage sludge, this study provides information on the effect of these treatments on the nature of humic substances binding microaggregates.

Materials and methods

Soi/s

The surface soils (0-20 cm) used for this study were collected from Modena, Lamporecchio (Lucca), and Cremona, in North-Central Italy between mid-October and early November 1986. In each of the three locations, samples representing the control and treated soils, were collected. On dry matter basis, the treated Modena plots received 40 Mg ha -1 pig slurry (between 1976 and 1985), those of Lamporecchio, 200 Mg ha -1 aerobically digested sewage sludge (between 1977 and 1980), whereas 8.0 Mg h a -1 of cattle slurry were added to the Cremona soil for 7 years. Further details on characteristics of amendments and on soil spreading techniques as well as the effects on soil properties and produc- tivity were reported elsewhere (Pagliai et al., 1981; Spallacci and Boschi, 1984). These soils were chosen to provide a wide range of management practices and variations in aggregate stability. Some of their pertinent properties are given in Table 1. Particle size analysis was done

Table I. Some important characteristics of the unamended (U) and amended (A) soils

Soil property Modena Lamporecchio Cremona (Fluventic Xerochrept) (Typic Psammaquent) (Aquic Xerofluvent)

U A U A U A

Sand (%) 56.8 54.2 66.1 64.6 49.2 46.4 Silt (%) 25.1 24.7 19.3 22.1 29.9 36.0 Clay (%) 18.1 21.1 14.6 13.3 20.9 17.6 O.M. (%) 1.16 2.64 0.91 2.80 2.57 2.96 pH (1:2.5 H20 ) 7.6 7.4 5.8 6.9 6.2 6.8 CEC (meq/100g) 21.7 23.9 16.9 20.7 21.7 25.0 MWDW a 0.63 0.73 0.17 0.50 0.39 0.47

a Mean-weight diameter of water stable aggregates, a structural stability index.

by the pipette method following the Internation- al System for size separation, organic carbon was determined by the dichromate oxidation technique, pH was measured in 1:2.5 soil-water suspension, and CEC by the sodium acetate method. Mean weight diameter of water-stable aggregates was obtained by the procedure of Kemper and Chepil (1965).

Separation of microaggregates and stability determination

After air drying the soil samples at room tem- perature, they were passed through a 4 mm sieve. Two hundred and fifty grams of the <4 mm diameter aggregates were then placed on the uppermost of a nest of 6 sieves having diameters of 2000, 1000, 500, 250, 125, and 50/xm, respectively, and shaken mechanically for 10 minutes using a RETSCH (plane rotary) sieving mach- ine. The microsieve sizes (<250 ~m) were further sieved by hand and all materials passing a particular sieve were transferred to the next and the process repeated. For each soil sample, mechanical sieving for the 250 g of <4.0 mm aggregates was done 3 times and all fractions within each size bulked together and stored in plastic containers for further studies.

Three fractions of microaggregates (250-125, 125-50, and <50 p~m) were utilized for the work presented here. For each microaggregate size, the Dispersion Ratio (Middleton, 1930), com- puted as

DR = percent silt + clay in water dispersed samples x 100 percent silt + clay in calgon dispersed samples

was used as an indirect test of water stability. DR is an index of the ease with which the particles should be brought into suspension by the action of the rain or by runoff water. The higher the value of DR the more the tendency for the particles to disperse in water and hence the lower the stability. DR measurements were made in triplicates for each microaggregate with a standard deviation never exceeding 0.2. A linear correlation was calculated between DR values and content of organic matter and humic

Microaggregates stability and humic substances 29

substances. The organic matter content as well as particle size analysis of microaggregates were determined as described above for the whole soil.

Extraction of humic substances

A sample (100 g) of each microaggregates fraction was first washed with 0.1N HCI and then with water until the washings were chloride-free. Thereafter it was extracted with 0.5N NaOH under N 2 atmosphere following the procedure outlined by Stevenson (1982). The resulting pre- cipitated humic acids (HAs) were finally washed twice with 0.05N HC1 and the washings dis- carded. The fulvic acids (FAs) left in solution were concentrated by eluting through an XADB column. The adsorbed FAs were eluted with 0.1N NaOH and immediately brought to pH = 2 with 2N HC1. Both FAs and HAs were then transferred into dialysis bags (Spectrapores of 1000 MW and 3500 MW cut off, respectively), dialyzed against distilled water until chloride- free, freeze-dried, weighed, and stored in a des- siccator for further analyses. Average percent ash, C, N, and H was 22.5, 47.3, 3.7, 4.2, for HAs, and, 5.1, 41.1, 2.0, 4.8, for FAs, respectively.

Molecular size of humic substances

The molecular weight distribution of the humic acids was assessed by a low pressure gel permeation chromatography (GPC) using Sephacryl $200 as gel matrix with an upper MW exclusion limit of 250.000. The gel packing solution and the eluent was a 1M 2-amino-2-hydroxymethyl- 1,3-propanediol (TRIS) hydrochloride buffer at p H - - 9 (Swift and Posner, 1972). Samples for chromatography (20 mg) were also dissolved in the same buffer. The absorbance of 470 nm of column eluates was continuously determined in flowcells of a UV-Vis detector (ISCO UA-5) and the relative chromatogram was automatically drawn by the built in recorder. Further details on the column calibration and gel performance were reported elsewhere (Piccolo and Mirabella, 1987). Both HAs and FAs were analyzed for the E4/E 6 ratio to obtain further information on the molecular sizes of extracts (Chen et al., 1977).


Results

Whole soil

The different types of additions of organic matter applied to the three soils have changed their general characteristics (Table 1). Particle size analysis was within the experimental error in all soils passing from unamended (U) to the amended samples (A). Organic matter content was significantly larger than control for all amended soils although the extent of the increase varied with the soil.

The aggregate size distribution for the whole soil calculated by the mean-weight diameter (MWD) (Kemper and Chepil, 1965), is also affected by the organic waste additions. The soil of Lamporecchio, which showed the least MWD, increased aggregate size distribution by 190% after 200 Mg ha -1 of sewage sludge whereas the aggregates of Cremona soil of intermediate MWD, revealed a 20% improvement after 8 Mg

ha-1 of cattle slurry. The aggregates of Modena soil with the highest original MWD were improved by only 16% even after 40 Mg h a -1 of pig slurry.

Microaggregates

Percent distribution of microaggregates in each soil before and after amendments is reported in Table 2. In the Modena and Cremona soils both the amounts of total microaggregates and of each microaggregate decrease with amendments whereas the Lamporecchio soil showed a percent increase for lower size microaggregates. The particle size distribution of microaggregates (Table 2) was different from that of the whole soil. For the unamended samples in both Modena and Lamporecchio soil, percent sand was higher than whole soil in the 250-125 and 125-50 /xm microaggregate sizes and smaller in the <50 ~m fraction. Conversely, per cent silt and clay were lower in the first two microaggregates but higher

Table 2. Percent distribution in soil (D), particle size distribution, Dispersion Ratio (DR), organic matter and humic substances contents of microaggregates

Soils/ Aggregate D Sand Silt Clay DR OM HA c FA c HA:FA Treatments a sizes b (%) (%) (%) (%) (%) (%) (%) (%)

Mu 1 13.4 65.5 14.4 20.1 37.4 1.45 19.3 1.70 11.4 2 7.1 66.6 14.6 18.8 49.5 1.57 12.7 1.68 7.6 3 3.8 49.4 26.1 24.5 51.4 1.90 13.0 1.40 9.3

Ma

Lu

La

Cu

Ca

1 8.3 64.4 16.0 19.6 28.5 2.28 32.3 1.84 17.6 2 5.5 62.3 17.9 19.8 44.2 2.21 18.8 1.32 14.3 3 3.4 51.8 25.3 22.9 43.2 2.38 16.8 1.44 11.7

1 12.2 72.7 13.8 13.5 39.8 0.71 50.7 4.23 12.0 2 8.1 76.6 10.8 12.6 21.1 0.62 85.9 5.16 16.6 3 7.2 56.7 27.2 16.0 64.0 0.84 43.2 5.67 7.6

1 10.4 77.5 11.4 11.1 64.9 1.90 36.5 3.51 10.4 2 9.1 78.8 9.8 11.4 68.9 1.81 33.2 3.69 9.0 3 8.8 62.3 25.3 12.4 54.1 2.26 23.5 2.94 8.0

1 3.8 48.8 31.4 19.8 25.6 2.31 46.3 5.87 7.9 2 2.6 60.0 23.2 16.8 62.8 2.24 33.8 6.35 5.3 3 4.7 44.7 38.4 16.9 31.6 1.60 43.8 8.91 4.9

1 2.9 48.6 31.1 20.3 75.1 3.36 28.9 5.38 5.4 2 2.1 59.5 23.4 17.1 68.2 2.19 40.1 7.16 5.6 3 3.3 51.4 32.2 16.4 19.4 1.71 49.5 9.48 5.2

a M = Modena; L = Lamporecchio; C = Cremona; U = Unamended; A = Amended. b 1 = 250--125 /xm; 2 = 125-50/,~m; 3 = <50/xm. ° Expressed as percent of organic matter content.

in the last fraction. For Cremona soil, the changes were similar except for the 250-125/xm aggregate size that gave a texture distribution comparable to the unamended whole soil and for the <50 /zm aggregates which showed a lower percent clay. A similar texture variation in re- spect to the amended whole soil were found for microaggregates of amended samples.

The organic waste additions to the soils pro- duced <50/xm aggregates richer in percent sand but poorer in silt and clay for all soils. The amendment generally decreased percent clay in each microaggregate fraction for Cremona soil. Percent silt in the 250-125 and 125-50/xm aggregates was increased for Modena and decreased for Lamporecchio whereas it remained practically unchanged for Cremona.

Organic matter, humic substances and aggregate stability"

For all three microaggregate fractions organic matter (Table 2) increased significantly when passing from unamended to amended samples in both Modena and Lamporecchio soil whereas it was only the 250-125 ~m fraction of Cremona soil that showed a significant increase. More- over, for the soils of Modena and Lamporecchio, the organic matter content was generally enhanced with the reduction of microaggregate size. Conversely, an organic matter decrease ac- companied the lowering of aggregate size in Cremona soil.

HA content (Table 2) as percent of total organic matter was enhanced in each microaggregate size of Modena soil by additions of pig slurry. Nevertheless, percent HA was progressively reduced with decreasing aggregate size in


amended samples. HA content also decreased with aggregate size in the amended Lamporec- chio soil but it was always lower than that found for the unamended samples. Conversely, Cre- mona soil showed a definite enhancement of percent HA with decreasing size of amended fractions. In the case of FA, the differences among aggregate size fractions and between treated and untreated samples were less evident than for HA.

Water aggregate stability (inversely related to Dispersion Ratio) was significantly increased in each microaggregate fraction of Modena soil following organic waste additions. However, aggregate stability tended to decrease going towards lower microaggregate size in both treated and untreated soils. For Lamporecchio soil, sewage sludge additions decreased aggregate stability of the 250-125 and 125-50 ~m size aggregates but somewhat enhanced that of the <50 /zm fraction. A similar behaviour was shown by the microaggregates of Cremona soil, where stability improvement resulted only in the <50 /~m fraction.

The Dispersion Ratio obtained for each microaggregate was correlated with organic matter content and humic substances values (Table 3). No correlation was found between Dispersion Ratio and percent organic matter whereas a significant relation was evident with HA content of microaggregates for all soils. The negative sign of the correlation coefficients is due to the inverse relationship existing between Dispersion Ratio and water aggregate stability. No significant correlation resulted with percent FA alone whereas, when this is added to percent HA, the correlation is highly significant for Modena soil and significant for Lamporecchio and Cremona

Table 3. Correlation coefficients between dispersion ratios and organic mat ter const i tuents of the microaggregates of different soils

Soils Correlat ion coefficient (r)

0 .M H A ~ FA a HS a H A : F A (%) (%) (%) (%)

Modena - 0 , 2 7 0 Ns -0 ,952"* - 0 . 6 6 3 s s -0 .957" . . . . 0.752" Lamporecchio 0.622ss -0 .848" - 0 . 3 3 8 s s -0 .838" -0 .883" Cremona 0.7049 Ns - 0 . 9 1 1 ' 0.243 r~s -0 .902* -0 .296 Ns

H A = Humic acids, F A = Fulvic acids, HS = Humic substances ( H A + FA). * Significant at P~<0.05, ** Significant at P~<0.01, NS Not significant.


Table 4. E J E 6 ratios of humic (HA) and fulvic (FA) extracts from microaggregates and positive ( + ) , negative ( - ) , and nil (0) changes from unamended to amended soils

Soil a Aggregate H A FA size b

U A Change U A Change

M 1 2.4 2.9 ( + ) 18.0 10.9 ( - ) 2 2.3 3.8 ( + ) 7.4 7.7 (0) 3 2.4 3.7 ( + ) 9.7 9.0 ( - )

1 2.5 2.6 (0) 9.3 7.8 ( - ) 2 2.4 1.8 ( - ) 9.8 7.8 ( - ) 3 3.1 3.3 (0) 7.4 11.2 ( + )

1 3.3 3.2 (0) 14.3 12.5 ( - ) 2 3.5 3.5 (0) 13.1 13.2 (0) 3 3.2 2.9 (0) 11.4 22.3 (+)

a M = Modena; L = Lamporecchio; C = Cremona; U = Unamended ; A = Amended. b 1 = 250-125 /zm; 2 = 125-50 /~m; 3 = < 5 0 / ~ m .

soil. The HA/FA ratio was also significantly correlated with aggregate stability of microaggregates of Modena and Lamporecchio soil.

Molecular size and distribution of humic extracts

The E4/E 6 ratio and its changes from unamended to amended samples for humic substances extracted from the microaggregate fractions are shown in Table 4. Only in Modena soil, the E4/E 6 ratio of HAs was found to increase after additions of organic waste, suggesting a reduction in molecular size in the treated samples,

whereas it remained generally constant for the two other soils. Conversely, in most aggregates, FAs, the lighter molecular weight humic material, appeared to have gained molecular complexity since E4/E 6 generally decreased with amendments, except for a significant increase in the <50 /xm aggregate of both Lamporecchio and Cremona.

Additional information was provided by the molecular weight distributions of the HA extracts by gel permeation chromatography (Fig- ures 1, 2 and 3). All chromatograms showed two peaks. The first peak corresponded to the high

0.11 0.1

E

,~ 0.08

=" 0.05

~. 0.02

Mu

~ . ~ . . - ~:: , ' . ..... • • ...%%

40 Vo 80 120 160 ELUTION VOLUME (ml)

0.2

E

o ,-- 014 .

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" I" ...... -,- "~i}'. ....~.%. ~ "",.., .. -..%. ~ '..%.% :

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Fig. 1. Gel Permeation Chromatography of humic acids extracted from microaggregates of Modena (M) sol. U = unamended; A = amended. Aggregate size: 2 5 0 - 1 2 5 / z m ( ); 125-50 g m ( . . . . . ); < 5 0 / ~ m ( . . . . . ).


0.1

= 0.08 f,,,,,,.

i L l

~ 0.05 Z

~ 0.02

+,,

o" ;

" " LU : "...

40 Vo 80 120 160 ELUTIOH VOLUME (ml)

0.2

- 0.16 LA r,,,,,,

w i t : 0.1 l~ | ; . ,++

~ it . . ' ° ° °'" %°%.

= \ _ , ~ , - . . . 0.06 ",~".. ,,., v "::.".

40 Vo 80 120 160 ELUTIOH VOLUME (ml)

Fig. 2. Gel Permeat ion Chromatography of humic acids extracted from microaggregates of Lamporecchio (L) soil. U = unamended; A = a m e n d e d . Aggregate size: 250-125 ~ m ( ); 125-50/ . tm ( . . . . . ); <50 ~ m ( . . . . . ).

molecular weight fraction eluted at the column void volume whereas the second peak repre- sented the low molecular weight fraction which was able to diffuse through gel pores. Molecular weight distribution of HA extracts from microaggregates revealed some differences among unamended soils. Mu (Fig. 1) showed a sharp and prominent first peak whereas Lu (Fig. 2) had almost no first peak but a broader second peak. Cu (Fig. 3) was placed in an intermediate situation since both peaks were prominent, the second higher than the first one, and reached an absorbance rather higher than Mu and Lu especially for the 125-50 and <50 /zm fractions.

The distribution chromatograms changed con- siderably for the amended samples. The HA extracts from all microaggregates showed a definite increase of the first peak as compared to the diffused peak. Furthermore, in all soils the <50 /xm aggregates revealed the highest increase in the first peak followed, in order, by the 125-50 and 250-125 /xm size aggregates. The absorbance gain showed by the first peak was not compensated by a comparable absorbance reduction of the diffused peak. The molecular weight distribution of fulvic acids were not reported here because there was no significant differences among microaggregates and between treated and untreated samples.

0.2

[ ,,-,, cu 0.14 l- ,',,, ,' ."".;,,

40 Vo 80 120 160 ELUTION VOLUME (ml)

0.2

O k . ' " ' .

0.14~ " " ~ ~ CA 0.1

~ 0.06 " ''.,

0.02 "'-', "". .- .~.~.

40Vo 80 120 160 ELUTION VOLUME (ml)

Fig. 3. Gel Permeat ion Chromatography of humic acids extracted from microaggregates of Cremona (C) soil. U = unamended, A = amended+ Aggregate size: 250 -125 /zm ( ); 125-50 t~m ( . . . . . ); <50 txm ( . . . . . ).


Discussion

Microaggregates

The 250-20/zm aggregate size fraction may play an essential role in the process of soil water erosion since it is stable against disruption by rapid wetting and agricultural practices (Tisdall and Oades, 1982). Macroaggregates (>250/zm) are more sensibly dependent on soil management practices reflecting the labile nature of the components responsible for their water stability. These are the transient binding agents such as the rapidly degradable soil polysaccharides (Fos- ter, 1978; Tisdall and Oades, 1979), and the temporary binding agents, of relatively higher longevity, such as roots and hyphae and some soil fungi (Foster and Martin, 1981; Oades, 1984). Additions of energy material to soils such as organic waste promote a buildup of large populations of microorganisms (Shields and Paul, 1973; Stevenson, 1982). The biomolecules excreted by microorganisms, such as polysaccharides and enzymes, are either further de- graded into smaller components (Martin, and Haider, 1971), blocked by complexation with metal ions and clay minerals (Brown and Lester, 1979; Martin et al., 1965), or incorporated into soil humic substances (Stevenson, 1982). The ultimate fate is to become part of the resistant organic matter (McGill et al., 1973, 1981) in organo-mineral complexes ( L a d d e t al., 1981) resulting in a considerable increase in soil aggregate stability (Griffith and Burns, 1972). In this way normally transient, labile compounds are stabilized into humic molecules resistant to further microbial degradation even after prolonged cultivation (Tiessen and Stewart, 1983). This heterogeneous structure represents the persistent binding agent for the <250/zm aggregates (Ed- wards and Bremner, 1967; Theng, 1979; Tisdall and Oades, 1982). The stability of microaggregates to dispersing factors (raindrop impact and exchangeable ions) is a prerequisite for the formation of relatively more stable soil macroaggregates.

The separation of the <250 /xm aggregates into groups of microaggregates which are not strictly silt and clay, have been rarely performed (Dormaar, 1983). Microaggregate fractions of 250-125, 125-50 and <50 /zm sizes are easily

obtained by dry sieving whereas the achievement of a 50-20 /zm size fraction implies methods destructive to the particle binding arrangement such as ultrasonic vibrations (Hamblin, 1977; Watson, 1971).

The effect of the organic waste amendment on the particle size distribution of microaggregates was to decrease the amount of microaggregates in soils (except Lamporecchio) and to lower the percent silt and clay and enhance percent sand in the <50/xm size fraction in all soils. This indicates that the organic compounds added to soils have linked together the fine particles promoting the formation of stable aggregates of higher dimensions which should be more resistant to dispersing factors. For Lamporecchio soil, a similar aggregation phenomenon was also shown for the 250-125, and 125-50 /~m size fractions. This may be attributed to the large percent increase in organic matter after amendments that may have counterbalanced the low clay content of this soil (Table 1). These results appear to support the report of Tisdall and Oades (1982) who assigned to persistent organic materials the cementing action in the 250-20/~m aggregate range.

Humic substances and aggregate stability

The results obtained in this study suggest that microaggregate stability correlates with the relative content of humic substances more than with any other component of soil organic matter. It is the percent humic acids that showed a parallel trend with the stability of different microaggregates. The latter decreased when the former was reduced. The same trend was followed by the sum of HA and FA whereas FA alone were not significantly related to aggregate stability. This is in line with other findings (Shanmuganathan and Oades, 1983) reporting that FA rather increase clay dispersibility. The fact that total organic matter content of microaggregates did not show a significant correlation with aggregate stability suggests that other organic constituents are not responsible for the stabilization. This assump- tions is further strengthened by the observation that, despite the increase of total organic matter in microaggregates of smaller size, the aggregate stability showed in most cases a decrease rather than an increase (Table 2). A similar effect was reported by Chaney and Swift (1986a and b) who

compared the capacity of humic substances and polysaccharides to form aggregates. They found that extracellular polysaccharides were less effective than humic substances in a long term stabilization of aggregates.

The effect of the organic waste amendments on the water aggregate stability of the three selected microaggregates varied with soils probably reflecting the different composition of added organic waste. However, a decrease in Dispersion Ratio was observed for the <50 tzm microaggregates. A concomitant enhancement of the absolute HA content with amendments was found in all size fractions of all soils (Table 2).

Molecular size and aggregate stability

The molecular weight distribution of the humic extracts represents a further characterization of the humic components responsible for microaggregate stability. Dell'Agnola and Ferrari (1971) separated in different soils, by a wet sieving mechanical technique, aggregates of >250 /zm size from those of <250 txm size. They defined the former as stable aggregates, and the latter as unstable aggregates. It was found that, using a Sephadex permeation chromatography, humic materials of molecular weight >100,000 were in the stable aggregates whereas humus with molecular weight <100,000 was predominant in the less stable aggregates. Chakraborty et al. (1979) reported that, by Sephadex gel filtration, smaller sized aggregates (<200 /xm) contained higher proportion of high molecular weight HA. Chaney and Swift (1984), though based only on the correlation between amount of extracts and types of extractant, concluded that, in macroag- gregate fractions of different soils, the less oxidized, higher molecular weight humic materials are more important in the process of aggregate stabilization than the lower molecular weight more oxidized humic substances.

In this work, for the unamended samples of each soil the initial structural stability (MWDW in Table 1) was reflected in the molecular weight distribution (Mu, Lu, and Cu in Figures 1-3) of humus extracted from microaggregates. In fact, for all microaggregates, the height of the peak at the void volume representing humus of high molecular weight (>250,000) was largely predominant over the second peak (<250,000) for


the most stable soil (Mu), negligible for the most fragile soil (Lu) and slightly lower than the diffused peak for the soil (Cu) with an intermediate aggregate size distribution. The difference in peak absorbance among the three microaggregates in each unamended soil was variable although the fractions of lower size (125-50 and <50 t~m) generally showed for both peaks a higher absorbanee than for the 250-125 ~m size fraction (Figures 1-3). This suggests that humic extracts of lower size microaggregates contain chromophore groups in either high number or in a large degree of conjugation (Stevenson, 1982). After amendments, the first peak (>250,000) was noticeably enhanced regardless of the type of added organic material (Ma, La, Ca in Figures 1-3). Furthermore, for all soils this high molecular weight peak showed an absorbance increase as the microaggregate size decreased. The <50 ~m size fraction had the highest absorbance. The second peak (<250,000) did not change significantly with amendments except for a slight increase in some size fractions (Figures 2 and 3). These findings demonstrate that additions of organic waste to soils enriched the high molecular weight fraction of humic substances in microaggregates rather than the low molecular weight fraction and that the <50 t~m size aggregate was mostly benefited. This confirms the reported literature works which examined microaggregates as a whole and further evidentiates that humic substances of higher degree of condensation (i.e.: high molecular weight) are increasingly present in lower size microaggregates.

The absorbance increase revealed by the high molecular weight peak for the <50 p~m aggregates is invariably coupled to a reduction of Dispersion Ratio (Table 2) and, thus, to an increase of aggregate stability when passing from unamended to amended samples. This indicates that the microaggregate enrichment in high molecular weight compounds contributes to the stability of microaggregates. This is also true for other microaggregate sizes in Modena soil whereas less consistent were the results for the 250-125 and 125-50 t~m size aggregates of the two other soils.

The E4/E 6 ratio is traditionally employed to indicate differences in molecular sizes of humic extracts, and it is believed to be inversely related to the molecular weight (Chen et al., 1977). The


values found for the E 4 / E 6 ratios of humic acids extracted from microaggregates did not show the same trend evidentiated by the reported molecular weight distributions. In most cases, the Ea/E 6 ratio failed to appreciate a significant change in the molecular arrangement of HAs after addition of organic waste. When the E n / E 6 results indi- cated a change in complexity, as for Modena soil, this was in contrast with gel permeation chromatograms which suggested, instead, a molecular weight enhancement. Piccolo (1988) showed the unreliability of E a / E 6 m e a s u r e m e n t s

to assess humus molecular sizes when humic materials are not thoroughly purified. The results shown here confirm that the E a / E 6 ratio for unpurified materials has limited value for the evaluation of molecular sizes of extracts.

This study shows that regular use of organic waste is capable of increasing the stability of all microaggregates (<250 /zm) of a fragile soil (Lamporecchio) and, at least, that of the lower size microaggregate (<50/~m) of the more stable soils (Modena and Cremona). Furthermore, the persistent binding agents responsible for the microaggregation after amendments with organic waste are the humic acids and their molecular weight progressively increases in lower size microaggregates. Although such beneficial effect is a product of a long term biological process in soils, it may be expected that the larger the average molecular size of organic matter in waste material, the higher the capacity for aggregate stabilization. In fact, direct additions of high molecular weight HA to soils were found to be effective in aggregate stability (Piccolo and Mbagwu, 1989; Mbagwu and Piccolo, 1989). No data are yet available, to our knowledge, on the molecular dimensions of the organic waste usual- ly employed in soil management. Information on the average molecular weight of organic matter in wastes to be used on agricultural soils would prove useful for an improved management of structurally fragile soils.

Acknowledgements

A Visiting Fellowship awarded to the second author by the International Centre for Theoreti- cal Physics (ICTP) Trieste, Italy, under the

"Training and Research in Italian Laboratories Programme".

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