Chemistry and micromorphology of aggregation in earthworm casts

Geoderma, 45 (1989) 357-374 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

357

Chemistry and Micromorphology of Aggregation in Earthworm Casts* M.J. SHIPITALO and R. PROTZ

USDA ARS, North Appalachian Experimental Watershed, P.O. Box 478, Coshocton, OH 43812 (U.S.A.) University of Guelph, Department of Land Resource Science, Guelph, Ont. NIG 2W1 (Canada) (Received August 3, 1988; accepted after revision April 10, 1989)

ABSTRACT Shipitalo, M.J. and Protz, R., 1989. Chemistry and micromorphology of aggregation in earthworm casts. Geoderma, 45: 357-374. The mechanisms by which earthworms produce and stabilize soil aggregates are not well understood yet this information is necessary before management practices that promote the beneficial aspects of their activity can be devised. Therefore, selective chemical pretreatments and micromorphological observations were used to investigate the nature of aggregate formation and stabilization in worm casts. Passage of soil through worms disrupted pre-existing microaggregates due to breakage of some bonds of the water and cation bridge type; however, incorporated organic debris fragments became plasma encrusted and served as nuclei for new aggregates. In excreted pellets, aging and drying facilitated close approach and bonding of plant and microbial polysaccharides and other organic compounds associated with the organic fragments to clay, thereby stabilizing the new microaggregates. In the soil material investigated, these bonds consisted predominantly of clay-polyvalent cation-organic matter (C-P-OM) linkages involving calcium when the worms were provided alfalfa- or corn-leaf diets.

INTRODUCTION

Earthworms have long been considered important in the development and maintenance of desirable physical properties of soils. The mechanisms by which earthworms improve soil structure are not well understood, however, and this information is required before management practices that optimize their beneficial effects can be developed (Syers and Springett, 1984). When we investigated factors influencing the dispersibility of clay in worm casts, we found that earthworms disrupt existing aggregates in the process of forming new ones (Shipitalo and Protz, 1988). For stabilization to occur, incorporation of or*Contribution of the University of Guelph, Department of Land Resource Science.

0016-7061/89/$03.50

© 1989 Elsevier Science Publishers B.V.

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M.J. SHIPITALO AND R. PROTZ

ganic debris into casts was essential, and aging or drying was necessary for incorporated organic matter to be effective in reducing dispersibility. Unfortunately, it is difficult to infer mechanisms by which organic matter stabilizes casts from analyses of earthworm diets or organic matter in casts because only a portion of the ingested material contributes to aggregation. In addition, the manner in which the active fraction of the organic matter is distributed is probably more important than is the total amount present. The reaction of soil aggregates to selective pretreatments can be used to infer the chemical nature of bonds and bonding materials, and micromorphological techniques can be used to ascertain the distribution of organic materials. Our objectives, therefore, were to investigate organo-mineral bonding and the distribution of organic materials in worm casts using these techniques and to develop a model for aggregate formation and stabilization. EXPERIMENTAL

PROCEDURES

Earthworm cultures were maintained in pots containing three specimens of Lumbricus terrestris L. and 4 kg of air-dried soil material held at 25% gravimetric moisture content. The sieved soil material used in the pots was collected from the Ap horizon of a coarse-loamy, mixed, mesic, Entic Hapludoll (Soil Survey Staff, 1975) at the University of Guelph corn tillage plots in Elora, Ontario, Canada. Organic carbon, clay, and sand contents of the < 2 mm-fraction were 2.14%, 13.2% and 18.2%, respectively. Soil pH was 7.0 and vermiculite and clay mica were the predominate clay minerals in the < 2-Ftm fraction. Diets provided to the worms consisted of alfalfa (Medicago sativa L. ) leaves, corn (Zea mays L.) leaves, or no-food. The cultures and pots containing soil material but no worms or food were kept in a darkened growth chamber maintained at 15 °C and 90% relative humidity. Additional information concerning worm, food, and soil material preparation is reported elsewhere (Shipitalo et al., 1988). Analysis of the plant materials was performed using the methods of Thomas et al. (1967). Surface casts from the cultures and soil samples from the controls without worms or food were obtained and immediately allowed to air dry at room temperature or were aged moist at 15 ° C for 0, 32, or 64 days. After drying or aging, individual casts and soil samples were analyzed for dispersibility, clay, sand, and organic carbon content using the methods given in Shipitalo and Protz (1988) or, prior to analysis, were subjected to one of the four chemical pretreatments outlined in Table I. Analyses were performed on four replicate casts for each diet-moisture-age-pretreatment combination and four soil samples for each moisture-age-pretreatment combination. A dispersion index (DI), defined as the ratio of percent clay suspended in distilled water to that measured upon addition of Na-hexametaphosphate Na-carbonate, was used as a measure of aggregate stability.

359

CHEMISTRY AND MICROMORPHOLOGY OF AGGREGATIONIN EARTHWORM CASTS TABLE I

Chemical pretreatment procedures and types of organo-mineral complexes affected Pretreatment

Duration 6 h

chloride

borate

periodate

pyrophosphate

50 ml 0.02 M NaCl

50 mi

50 ml

50 ml

0.02 M NaC1 $

0.02 M NaI04 ~

0.025 M Na4Pz07 (pH 10.2)

drained

drained

drained drained

Duration 2 h

50 ml 0.01 M NaC1 ,,

drained and

Organo-mineral complexes

affected

50 ml

50 ml

0.05 M Na2B40v (pH 9.6) ~

0.05 M NazB407 (pH 9.6)

drained

drained

suctioned

and suctioned

and suctioned

Negatively charged organic matter bonded to clay via water and cation bridges

Same as chloride treatment plus the effect of high pH

Same as borate treatment plus bonding of neutral, neg. andpos, charged polysaccharides

and suctioned -

Same as borate

treatment plus organic matter bonded via coordination

complexes

Pretreatment was accomplished by placing individual casts or soil samples in the upper funnel of 0.45 Ftm membrane filtration units with plugged funnels. After the prescribed soaking period, the plugs were removed and vacuum applied, allowing the pretreatment solutions to pass through the filters into the lower compartments. The extracted solutions were subsequently analyzed for Ca, K and Mg contents by atomic absorption flame spectrophotometry. The casts and soil samples were then transferred to bottles for dispersion analysis. Various opinions exist concerning the specificity of the periodate pretreatment. A 6-hr treatment with Na-periodate reportedly does not result in the complete destruction of soil carbohydrate (Cheshire et al., 1983, 1984). Oades (1984), however, questions the selectivity of periodate pretreatment when either stronger solutions or longer treatment periods are used as Cheshire et al. (1983, 1984) recommend. Action of all the pretreatments is probably dependent on chemical and physical properties of the materials investigated. In addition, pretreatment may rupture bonds without extracting or destroying the bonding materials. The value of the various pretreatments lies in the fact that comparative differences in response among samples can be used to infer differences in the nature of the organo-mineral complexes bonding aggregates

360

M.J. SHIPITALO AND R. PROTZ

together. Nevertheless, Tisdall and Oades ( 1982 ) indicate that the most convincing evidence that polysaccharides stabilize aggregates comes from the use of periodate as a selective oxidant and measurement of subsequent losses in aggregate stability. Thin sections were prepared from air-dried casts impregnated under vacuum with 3-hydroxy-butyl methylmethacrylate and cured with gamma radiation. Thin sections suitable for histochemical staining and electron microscopy were prepared from air-dried casts fixed with 2 % glutaraldehyde in Sorensen's phosphate buffer at the natural pH of the soil (pH = 7) as recommended by Foster et al. (1983, p. 10). These samples were dehydrated with ethanol and propylene oxide and embedded with an epoxy resin, Epon 812, following the procedure of Luft (1961). Fixation with aldehydes cross-links organics thereby making them less susceptible to solubilization and rearrangement during dehydration and embedding (Foster, 1978). Features observed in thin section were described using the terminology of Brewer {1976). Polysaccharide distribution was determined by staining Epon embedded sections using Periodic Acid Schifi's (PAS) reaction (O'Brien and McCully, 1981, p. 6.83-6.84) for examination using light microscopy, whereas the Thiery reaction (periodic acid-thiocarbohydrazide-silver proteinate) was used for examination by electron microscopy (Lewis and Knight, 1977, p. 102 ). A positive reaction to PAS is indicated by reddish-purple staining, whereas deposition of metallic silver indicates the presence of Thiery-reactive material. Both techniques stain similar materials and rely on conversion of polysaccharides to aldehydes by periodic acid (Lewis and Knight, 1977, p. 97 and 102 ). Dimedone (5,5-dimethylcyclohexane-1,3-dione ) was used to block pre-existing aldehydes and those introduced during fixation to ensure specificity for aldehydes produced by periodic acid. An additional feature of these techniques is that both rely on reaction with periodic acid and should therefore stain materials susceptible to Na-periodate pretreatment. Data analyses were performed using the Statistical Analysis System (SAS Inst., 1982). A 5% probability level for significance was chosen as the minimum acceptable for all analyses and multiple comparisons were made using Tukey's method. RESULTS

Chemistry of aggregation Air-dried casts The effect of diet on dispersibility was assessed by comparing DI's among diets within pretreatments and by examining relationships between DI and cast organic carbon content (Tables II, and III). The degree of aggregation in untreated, air-dried casts was greatest when L. terrestris were given a corn-leaf

CHEMISTRY AND MICROMORPHOLOGY OF AGGREGATIONIN EARTHWORMCASTS

361

T A B L E II Comparison of mean DI's for dried casts and soil samples Diet or material

No food Dried soil samples Corn leaves Alfalfa leaves

Pretreatment untreated

chloride

borate

periodate

pyrophosphate

0.44a (w)*' 0.45a(w)

0.81a(x) 0.79ab(x)

0.82a(x) 0.78ab(x)

0.84a(y) 0.83ab(y)

0.91a(z) 0.91a(z)

0.30c(w)

0.73b(x)

0.78ab (xy)

0.81b(y)

0.92a(z)

0.38b (w)

0.59c (x)

0.76b (y)

0.78c (y)

0.91 a ( z )

*'Means in the same column with no letters in common ( a - c ) a n d means in the same row with no letters in c o m m o n (w-z) are significantly different P ~ 0.05. T A B L E III Relationships between D! a n d organic carbon content for dried casts from L. terrestris fed alfalfa or corn leaves Pretreatment

Relationship between DI and organic carbon

r2

P

Alfalfa leaves Untreated Chloride Borate Periodate Pyrophosphate

DI= DI= DI = DI= DI =

0.45 0.67 0.68 0.38 0.001

0.33 0.18 0.18 0.38 0.97

Corn leaves Untreated Chloride Borate Periodate Pyrophosphate

DI= 0.02(% OC) +0.22 D I = - 0 . 0 5 ( % OC) +0.91 DI= 0.06(% O C ) + 0 . 5 2 DI= 0.01(% O C ) + 0 . 7 5 DI= 0.03(% O C ) + 0 . 7 8

0.02 0.61 0.84 0.80 0.97

0.88 0.22 0.08 0.11 0.01

- 0 . 1 8 (% - 0 . 1 6 (% - 0.08 ( % - 0 . 1 1 (% 0.002 (%

OC) + 1.02 OC)+1.17 OC ) + 1.05 OC) + 1.14 OC ) + 0.90

diet and somewhat less with an alfalfa-leaf diet (Table II). Mean DI of casts produced when no food was provided was similar to that for uningested, airdried soil samples. Pretreatment with chloride resulted in a mean DI for casts produced by worms fed corn leaves that was significantly higher than that for casts produced when alfalfa leaves were fed (Table II). This indicated that bonds attributable to ingestion of corn leaves were more sensitive to chloride pretreatment than those attributable to incorporation of alfalfa-leaf residue into casts. The inverse relationship between DI and organic carbon observed

362

M.J. SHIPITALO AND R. PROTZ

when casts from worms fed corn leaves were pretreated with chloride, (Table III) and the fact that DI was significantly less than that of casts produced when on the no-food diet (Table II) suggested, however, that this pretreatment did not entirely negate the bonding imparted by corn-leaf residue. Pretreatment with borate, when compared to pretreatment with chloride, caused a large increase in mean DI of casts produced by worms fed alfalfa leaves (Table II). Mean DI of these casts was not significantly less than that of air-dried, borate pretreated soil samples but was significantly less than that of casts produced when no food was provided (Table II). An inverse relationship between DI and organic carbon also indicated that borate pretreatment did not completely eliminate bonding attributable to alfalfa-leaf residue (Table III). Borate pretreatment did, however, negate the beneficial effects of the corn-leaf residue as suggested by the positive relationship between DI and organic carbon and by the lack of significant differences among mean DI's of airdried soil samples and casts produced by worms on the no-food and corn-leaf diets (Tables II, III). Periodate pretreatment produced results similar to those observed with borate. When pretreated with periodate, casts from worms on the alfalfa-leaf diet had a significantly lower mean DI than all other samples and an inverse relationship between DI and organic carbon was observed (Tables II, III). Only upon pyrophosphate pretreatment did mean DI of casts from worms fed alfalfa equal the DI's of all other samples (Table II). In addition, for casts produced by worms fed alfalfa leaves, pyrophosphate pretreatment resulted in a positive relationship between DI and organic carbon, whereas inverse relationships were observed with all other pretreatments (Table III). This indicated that a portion of the improved microaggregation attributable to bonds resulting from incorporation of alfalfa-leaf residue into casts was resistant to all pretreatments except pyrophosphate. All the pretreatment extracts from casts produced by worms on the alfalfaleaf and corn-leaf diets contained significantly more K + than did the extracts from dried soil samples and casts produced by worms receiving no food (Table IV). The source of the additional K + was probably the incorporated alfalfa and corn leaves which contained considerable amounts of K (Table V). Because alfalfa and corn leaves also contained Ca and Mg (Table V), mean Ca 2+ and Mg2+ removals by each pretreatment should have been greater for casts produced by worms fed alfalfa or corn leaves than for those provided no food and from dried soil samples. This, however, was not the case. The amount of Ca e+ removed by chloride and borate from casts produced by worms fed corn or, more evidently, alfalfa leaves, was much less than that removed from casts produced when worms received no food or from dried soil samples (Table IV). With periodate and pyrophosphate pretreatments, however, mean Ca 2+ removals from casts produced by worms fed corn leaves were greater than those from casts produced by worms not provided food and dried soil samples. The

CHEMISTRYANDMICROMORPHOLOGYOF AGGREGATIONIN EARTHWORMCASTS

363

TABLE IV Comparison of mean amounts of K, Ca and Mg extracted from dried casts and soil samples by each pretreatment

Pretreatment

Diet or material

chloride

borate

periodate

0.37a *l 0.30a 0.89b 1.08b

0.33a 0.29a 1.03b 1.15b

pyrophosphate

cmol (K +) kg -1 No food Dried soil samples Corn leaves Alfalfa leaves

0.38a 0.31a 0.95b 1.13b

0.21a 0.17a 0.74b 0.77b

cmol (1~2Ca 2+) kg -1 No food Dried soil samples Corn leaves Alfalfa leaves

8.7b 10.2a 7.4b 4.9c

8.5b 10.4a 7.0c 4.8d

5.6b 7.lab 9.3a 4.8b

9.5bc 12.3ab 14.0a 9.0c

2.0a 2.0a 3.8b 1.8a

1.8a 1.9a 3.9b 1.9a

1.6a 1.9a 5.3b 2.0a

2.3a 2.6a 7. lb 3.5a

cmol (1/2Mg 2+) kg--1 NO food Dried soil samples Corn leaves Alfalfa leaves

*1Means in the same column within cationic species with no letters in common are significantly different at P~< 0.05. TABLE V Chemical composition of the two food sources Material

N

P

K

Ca

Mg

Organic

(%)

(%)

(%)

(%)

(%)

C (%)

C/ N

Alfalfa leaves

4.20

0.60

2.67

4.96

0.64

42.0

I0.0

Corn leaves

2.45

0.44

1.58

2.30

1.11

43.5

17.8

mean amount of Ca 2+ solubilized by periodate pretreatment of casts produced by worms on the alfalfa-leaf diet was less but not significantly different from that removed from casts produced by worms offered no food or from dried soil samples. Similar results were obtained upon pyrophosphate pretreatment (Table IV). These results indicated that a portion of the Ca 2+ was not released unless the pretreatment disrupted bonding; hence Ca 2+ may have been involved in linking incorporated organic matter to clay. For all pretreatments the mean amount of Mg 2+ removed from casts pro-

364

M.J. SHIPITALO AND R. PROTZ

duced by worms on the corn-leaf diet was significantly greater than that removed from the other samples (Table IV). This suggested that a portion of the Mg 2+ associated with organic matter from the Mg-rich corn leaves (Table V) was removed by all pretreatments. For casts produced by worms provided alfalfa leaves, the amount of Mg 2+ removed when casts were pretreated with chloride or borate was similar to that removed from dried soil samples and casts from unfed worms but was slightly higher when pretreated with periodate or pyrophosphate (Table IV ). As with Ca 2+, these results suggested that Mg :+ may have served to link clay to organic matter.

Moist casts Untreated moist casts from L. terrestris, regardless of diet or age, were more dispersible than uningested moist soil samples (Table VI). Casts produced by worms fed alfalfa or corn leaves were less dispersible, however, than those from worms not provided food indicating that incorporated organic matter stabilized microaggregates within casts. Moist casts from worms fed corn and more evidently alfalfa leaves were less dispersible when pretreated with chloride than moist soil samples and casts produced by worms not provided food and the differences were more pronounced in aged casts than in unaged casts. This greater resistance to dispersion with increased age suggested that additional chloride-resistant linkages formed over time. When pretreated with borate, casts from worms on the alfalfa-leaf and corn-leaf diets were as dispersible as those produced by u n f e d worms when compared at 0 days but were significantly less dispersible when compared at 32 days. This indicated that some borate-resistant bonds formed upon aging. When pretreated with periodate or pyrophosphate, casts and moist soil samples of all ages were equally dispersible indicating that these pretreatments eliminated any bonding effect attributable to ingestion of alfalfa or corn leaves. Organic matter and polysaccharide distribution Micromorphological examinations indicated that plasma (colloidal mineral and organic material) distribution and its relationship to organic and mineral skeleton grains (material larger than colloidal size) were a function of diet provided to the worms (Figs. 1-4). In casts produced by unfed worms, mineral skeleton grains were embedded in a dense groundmass of plasma and only simple packing voids were present (Fig. 2 ). Thus the related distribution pattern was porphyroskelic. In contrast, plasma was concentrated in the vicinity of organic skeleton grains in casts produced by worms fed corn or alfalfa leaves and compound as well as simple packing voids were observed; hence the related distribution was intertextic (Figs. 1, 3). Two types of organic skeleton grains were present in casts produced by worms

CHEMISTRYAND MICROMORPHOLOGYOF AGGREGATIONIN EARTHWORMCASTS

365

T A B L E VI Comparison of mean DI's for moist casts and soil samples Diet or material

Age 0 days

32 days

64 days

0.76a (x) .1 0.58c (x) 0.66b (x) 0.62bc(y)

0.76a(x) n.d.** 0.65b ( x ) 0.63b(y)

0.68a (x) 0.57b(x) 0.63ab ( x ) 0.67a(x)

0.82a (x) 0.80a (x) 0.77a ( x ) 0.68b (x)

0.81a (x) n.d. 0.72b (y) 0.59c (y)

0.78a (x) 0.79a (x) 0.72b (y) 0.62c (y)

0.83ab (y) 0.80b (x) 0.82ab (x) 0.85a ( x )

0.85a (x) n.d. 0.78b ( x ) 0.77b (y)

0.79a (z) 0.80a (x) 0.78a (x) 0.78a (y)

0.87a(x) 0.86a (x) 0.87a (x) 0.90a (x)

0.86a(x) n.d. 0.86a (x) 0.89a (x)

0.85a(x) 0.85a ( x ) 0.88a (x) 0.88a (x)

0.94a ( x ) 0.93a (x) 0.95a (x) 0.94a(x)

0.93a (x) n.d. 0.94a(x) 0.93a(x)

0.93a ( x ) 0.92a ( x ) 0.93a (x) 0.93a(x)

Untreated No food Moist soil samples Corn leaves Alfalfa leaves

Chloride pretreated No food Moist soil samples Corn leaves Alfalfa leaves

Borate pretreated No food Moist soil samples Corn leaves Alfalfa leaves

Periodate pretreated No food Moist soil samples Corn leaves Alfalfa leaves

Pyrophosphate pretreated No food Moist soil samples Corn leaves Alfalfa leaves

*1Means in the same column within pretreatments with no letters in common (a-c) and means in the same row with no letters in common (x-z, x is most, z is least dispersible ) are significantly different at P~< 0.05. **n.d. = no data.

on the corn-leaf diet. Relatively large pieces of debris with intact cellular structures, identified as remnants of vascular bundles, can readily be observed in Figs. la and lc. A high lignin content probably made this tissue difficult to decompose and contributed to its strong autofluorescence (Fig. lc). The fluorescence from lignin and other phenolic compounds in plant cell walls is yellow when excited by blue light (O'Brien and McCully, 1981, p. 2.44). The vas-

366

M.J. SHIPITALO AND R. PROTZ

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CHEMISTRY AND MICROMORPHOLOGY OF AGGREGATION IN EARTHWORM CASTS

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cular bundles did not react with Periodic Acid Schiff's (PAS) stain and only limited staining was observed in the surrounding plasma. The other type of debris consisted of strands and clumps of organic material with no apparent cellular structure. This type of debris can be been scattered throughout Fig. la, though a thin strand can best be observed in the lower portion of Fig. lb. The non-cellular debris was probably comprised of the remains of the mesophyll and epidermis tissues that originally surrounded the vascular bundles and fungal hyphae that were observed in abundance on the leaves prior to ingestion. This debris reacted strongly to PAS and staining was observed in the surrounding plasma (Fig. ld), indicating that it contained polysaccharides and that plasma associated with it may have been more polysaccharide-rich than that associated with cellular debris. Concentration of plasma was more apparent in the vicinity of non-cellular debris than around remnants of vascular bundles (Fig. lb and c). Casts produced by worms on the alfalfa-leaf diet contained many short, elongated pieces of organic debris with preserved cellular structure (Fig. 3 ). This debris was comprised of petioles and veins of the alfalfa leaves and concentration of plasma adjacent to this material was readily apparent in thin sections (Fig. 3b). The cellular fragments generally exhibited a positive reaction with PAS stain. In addition, PAS-sensitive material appeared to be well interspersed in the soil matrix due to highly fragmented debris that could not be resolved or to decomposition products associated with the cellular organic fragments, mucilage. Oades (1984) postulates that mucilages are a product of the decomposition of organic matter and that they consist primarily of polysaccharides. Mucilages associated with root surfaces reportedly are stained by PAS (Oades, 1978). Although organic skeleton grains appeared important in initiating plasma aggregations, their distribution was not homogeneous within casts. This is illustrated in Fig. 3a which shows two welded pellets within a cast produced by a worm fed alfalfa leaves. One pellet contains little incorporated organic debris as indicated by its low emission of fluorescent light, whereas the other pellet contains considerable amounts. Close examination suggests that distribution of organic debris was quite variable even within pellets. Observation of sections stained using the Thiery reaction supported the polysaccharide distribution patterns inferred from examinations of PAS stained sections. Spot analysis using energy dispersive spectrometry confirmed the presence of Ag, hence Thiery-sensitive materials, in locations similar to those noted with PAS stain but Ag contents were too low to obtain elemental distribution maps. When samples were examined at high magnification using a scanning transmission electron microscope, Ag grains were observed on clay particles (Fig. 4), indicating intimate association of clay and polysaccharide.

CHEMISTRY AND MICROMORPHOLOGY OF AGGREGATIONIN EARTHWORM CASTS

369

Fig. 4. Thiery-stained cast from a worm fed corn leaves showing Ag deposits (bright spots) on clay platelets, S T E M backscatter electron image (7,666 × ).

DISCUSSION

Our results and observations suggested the model for aggregate formation in worm casts depicted in Fig. 5. In the first step, litter and soil material are ingested and intimately mixed. Litter is fragmented by the grazing activity of worms and a liquified soil-debris mixture forms in the anterior portions of the digestive tract. The organic debris is subject to further fragmentation by strong muscular contraction in the gizzard which grind the food with the aid of mineral particles (Edwards and Lofty, 1977, p. 22 ). Lignified components, such as vascular bundles, resist fragmentation and remain as relatively large, intact pieces, whereas less lignified and softer tissues are highly fragmented and become part of the soil plasma. Fungal hyphae present in the organic debris and ingested soil are also subject to fragmentation. Bal {1982, p. 165-166) also noted that little fragmentation occurs when litter which is difficult to decompose is supplied to worms. Physical breakdown of the tissue, combined with microbial activity and the action of digestive enzymes and secretions produced by the worm, results in the complete decomposition of some organic material as well as the release and formation of bonding agents. Continual trituration breaks many of the existing weak interparticle bonds but also brings domains of clay minerals in close association with newly formed or released bonding agents. In the intestine, flocculation or coagulation causes aggregations of organic matter-enriched plasma to form. Plasma also adheres to the surfaces of organic skeleton grains where concentrations of bonding materials may be present as layers of mucilage. Thus, resistant organic fragments become the foci of aggregate formation. At this point, however, bonding

370

M.J.SHIPITALOANDR.PROTZ ORGANIC MATTER

SOIL AGGREGATES

grazing initially fragments organic materials -- soil-organic debris slurry forms grinding action of gizzard further fragments debris lignified components maintain cellular structure -- soft tissues decompose entirely or are incorporated into the plasma -- binding substances are released or formed - -

- -

- -

EXCRETION -- organic matter enriched plasma coagulates -- resistant organic fragments become plasma encrusted -- pellets are formed

"~

~ / (

Mucilage layer

/ ~

. ~

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mixture-----~f pla~nt and microbial / polysaccharides and other ~anics

,

F e c a l

Pellet / . . . ~ ! ~ , ~11,\ [ urgan,c ~ ' ~ ' _ ~ / ~ X

~

Organic Matter G~iched Prasma

clay domains associated with organic polymers

Fig.5. Diagrammaticrepresentationof aggregateformationin earthwormcasts. between materials is relatively weak due to high water content and low moisture potential of the system. Pellets are excreted in this state. In excreted pellets, bonds between organic and mineral components are strengthened when these materials are brought into close association. This is encouraged by drying which dehydrates organic materials and can cause

CHEMISTRY AND MICROMORPHOLOGY OF AGGREGATION IN EARTHWORM CASTS

371

shrinkage. Aging also brings about closer association of mineral and organic materials in moist casts and promotes bond formation due to the process of thixotropic hardening which involves rearrangement of water films. Close association of clay domains with organic materials may slow decomposition, adding to bond longevity. Further decomposition of plasma-coated organic fragments may result in continued repl¢nishment of bonding agents to the surrounding plasma. Large organic fragments may link the microaggregates within pellets and the pellets within casts to form macroaggregates. Concepts similar to the one proposed above in which macroscopic organic materials serve as foci for aggregate formation have been put forth by several workers, mainly in reference to fungal hyphae or root hairs (Edwards and Bremner, 1967; Aspiras et al., 1971; Oades, 1978, 1984; Tisdall and Oades, 1979, 1982; Reid and Goss, 1981 ). The enmeshment of soil particles by fungal hyphae (Aspiras et al., 1971) or root hairs (Reid and Goss, 1981) reportedly results in only limited improvement in aggregate stability unless bonding of soil particles to their surfaces occurs. Tisdall and Oades (1979) found that some fungal hyphae produce layers of amorphous material, which they indicate may be a polysaccharide, to which clay particles were firmly attached. Therefore, Tisdall and Oades (1982) suggest that fragmentation of fungal hyphae could lead to formation of small aggregates. They also suggest that persistent bonding agent's are derived from resistant fragments of roots, hyphae and bacterial colonies which are surrounded by clay rather than vice versa. Organic material thus encapsulated is thought to be resistant to rapid decomposition because it is physically protected from attack by microorganisms (Edwards and Bremner, 1967; Tisdall and Oades, 1982; Oades, 1984). According to Oades (1978, 1984), mucilages form wherever organic debris is being decomposed and exist as immobile gels or fibrillae. In the case of earthworms, ingestion of soil and litter represents a mechanism whereby clay is brought into intimate association with decomposing, mucilage-coated, organic fragments which promotes formation of organic matter-cored microaggregates. The type and extent of bonding that occurs between mineral and organic materials in worm casts will depend on properties of the ingested organic debris and chemical and mineralogical properties of the soil materials. It is highly unlikely that a single class of compounds or mechanism of bonding is solely responsible for aggregate stabilization in any natural soil. Nevertheless, the chemical pretreatments did provide useful information as to the nature of the dominant organic components and bonding mechanisms responsible for enhanced aggregation in the casts (Fig. 6). Ingestion and mixing of moist soil probably disrupted some existing interparticle water and cation bridges. This was suggested by the observation that dispersibility of moist casts from unfed worms was greater than that of uningested moist soil samples but following chloride pretreatment the dispersibilities were similar (Table VI ). Restoration of aggregate stability in casts with

372

M.J.SHIPITALOANDR. PROTZ

WATER BRIDGES

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-

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c..O

\OH

c..O

~

O"-H20.-'Cal

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CATION BRIDGES

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COORDINATION COMPLEXES XO,,,Ca

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Illlllllllllll

IIIIIIII

BOND FORMATION - - clay unattached or loosely sorbed in fresh casts - - aging and drying promote bond formation - - negatively charged organic matter and C-P-OM linkages enhance microaggregate strength - - Ca and Mg cations link clay and organic matter

Fig. 6. Bonding mechanisms between clay and organic matter in casts produced by earthworms provided alfalfa or corn leaves.

incorporated alfalfa- or corn-leaf debris was predominantly the result of negatively charged organic compounds. All samples were equally dispersible following pretreatment with pyrophosphate, which does not affect bonding of positively charged and neutral organics. Thus, linkages consisting of clay-polyvalent cation-organic matter (C-P-OM) bonds appeared to be the dominant mechanism responsible for enhanced aggregate stability. The organic compounds involved in the C-P-OM bonds probably consisted of a complex mixture of plant and microbial polysaccharides as well as other organic polymers. Examination of stained thin sections indicated that polysaccharides were positionally available to contribute to bond formation. Pretreatment of dried casts with pyrophosphate, however, resulted in greater dispersibilities of casts produced by worms fed alfalfa or corn leaves, when compared to casts from unfed worms, than did pretreatment with periodate, indicating that materials other than polysaccharides also contributed to enhanced aggregation. In addition, pretreatment with periodate did not greatly increase dispersibility above that of borate pretreated samples, indicating that cationic and neutral polysaccharides did not play a major role in enhancing aggregate stability. Calcium, and to a lesser extent Mg, appear to have been the polyvalent cat-

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ions most involved in C - P - O M linkages formed with incorporated alfalfa- and corn-leaf debris. This was suggested by the fact that, in general, less Ca 2+ was solubilized by chloride or borate pretreatment of casts from worm fed alfalfa or corn leaves than from soil samples, although more K + was solubilized and the incorporated organic debris was a potential additional source for both Ca and K. Different responses to pretreatments following aging and drying indicated that differences in type and extent of C - P - O M linkages occurred among diets. In moist casts that had been aged, the bonds were generally chloride- and borate-sensitive which suggested that the linkages consisted predominantly of water and cation bridges for both food treatments. The improvements in aggregate stability were only slight, however, when compared to those imparted by drying which indicated that limited bond formation occurred when casts were kept moist. This is consistent with a C - P - O M mechanism because relatively little adsorption of anionic polymers occurs unless dehydration allows short range forces to become operative (Theng, 1982). Although greater bonding occurred in dried casts from worms fed corn leaves than in ages, moist casts, the bonds were chloride- or borate sensitive suggesting that water and cation bridges were still the predominant bond type. In dried casts from worms fed alfalfa leaves, the bonds were partially resistant to all pretreatments except pyrophosphate, indicating that a significant proportion consisted of coordination complexes. This suggested that the organic polymers derived from ingested alfalfa leaves either contained more anionic groups or that they are more closely spaced than those on the organic polymers derived from ingested corn leaves. Bonding occurred immediately in unaged dried casts which indicated that microbial activity was not necessary to produce bonding agents from the organic materials investigated. Post-depositional microbial activity may be necessary, however, to produce bonding agents from different types of organic debris. In addition, microbial activity should eventually alter or destroy the bonds attributable to ingested alfalfa or corn leaves. It is not known how differences in bond type might affect bond longevity.

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