Protection of soil carbon by microaggregates within earthworm casts

Soil Biology & Biochemistry 37 (2005) 251–258 www.elsevier.com/locate/soilbio

Protection of soil carbon by microaggregates within earthworm casts Heleen Bossuyta,*, Johan Sixb, Paul F. Hendrixa,c a Institute of Ecology, University of Georgia, Athens, GA 30602, USA Department of Agronomy and Range Science, University of California, Davis, CA 95616, USA c Department of Crop and Soil Sciences, University of Georgia, Athens, GA 30602, USA

b

Received 2 January 2003; received in revised form 18 February 2004; accepted 10 July 2004

Abstract Earthworms are known to play a role in aggregate formation and soil organic matter (SOM) protection. However, it is still unclear at what scale and how quickly earthworms manage to protect SOM. We investigated the effects of Aporrectodea caliginosa on aggregation and aggregate-associated C pools using 13C-labeled sorghum (Sorghum bicolor (L.) Moench) leaf residue. Two incubations were set up. The first incubation consisted of soil samples crushed !250 mm to break up all macroaggregates with three treatments: (i) control soil; (ii) soilC13Clabeled residue and (iii) soilC13C-labeled residueCearthworms. Earthworms were added after 8 d and 12 d (days) later, aggregate size distribution was measured together with total C and 13C in each aggregate fraction. A second incubation was made to assay protected versus unprotected total C and 13C from 21-d laboratory incubations of intact and crushed large (O2000 mm) and small (250–2000 mm) macroaggregates and microaggregates (53–250 mm). Eight different pools of aggregate-associated C were quantified: (1) and (2) unprotected C pools in large and small macroaggregates, (3) unprotected C pools in microaggregates, (4) and (5) protected C pools in large and small macroaggregates, (6) protected C pool in microaggregates, and (7) and (8) protected C pools in microaggregates within large and small macroaggregates. In the presence of earthworms, a higher proportion of large macroaggregates was newly formed and these aggregates contained more C and 13C compared to bulk soil. There were no significant differences between the samples with or without earthworms in the C pool-sizes protected by macroaggregates, microaggregates or microaggregates within small macroaggregates. However, in the presence of earthworms, the C protected by microaggregates within large macroaggregates was a significant pool and 22% of this C pool was newly added C. In conclusion, these results clearly indicate the direct involvement of earthworms in providing protection of soil C in microaggregates within large macroaggregates leading to a possible long-term stabilization of soil C. q 2004 Elsevier Ltd. All rights reserved. Keywords: Aggregation; Microaggregates; Carbon; Earthworms; Carbon protection

1. Introduction Soil aggregation has a great influence on the physical characteristics of the soil. Well-aggregated soils possess a larger pore space, a higher infiltration rate and better gaseous exchange between soil and atmosphere than poorlyaggregated soils, leading to enhanced microbial activity (Lynch and Bragg, 1985). Soil aggregation and soil organic matter (SOM) dynamics are closely linked. Aggregates are thought to play an important role in the physical protection * Corresponding author. Address: Laboratory for Soil and Water Management, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium. Tel.: C32 16 32 96 76; fax: C32 16 32 19 97. E-mail address: [email protected] (H. Bossuyt). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.07.035

of SOM and at the same time, SOM binds with mineral particles to form aggregates of different sizes (Tisdall and Oades, 1982). The preservation of SOM is desirable for land use since SOM is widely recognized as a key component in nutrient cycling. Furthermore, the retention of organic C in soil is becoming more important since the rise in atmospheric CO2 and global warming are recent concerns (Schlesinger, 1997). Earthworms are considered to improve soil aggregation and they are known to promote the cycling of nutrients (Lee and Foster, 1991; Edwards and Bohlen, 1996). They play a crucial role in the removal of plant litter and other organic materials from the soil surface and the incorporation of these organic materials into soil aggregates (Martin, 1991). Earthworms ingest organic matter and mix it with inorganic

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soil material. This mixture passes through their gut and is excreted as a cast, which contributes to soil aggregation. Casts occur mostly in the upper 0–20 cm of the soil (Lee and Foster, 1991) and contain more water-stable aggregates than surrounding soils (Shipitalo and Protz, 1988; Marinissen, 1994). The formation of water-stable macroaggregates (O250 mm) depends primarily on temporary binding agents (Tisdall and Oades, 1982). Earthworms play a role in the formation of these binding agents (Martin and Marinissen, 1993) through secretion of mucus in their gut. Microbial polysaccharides and other organic products in the casts may strengthen bonds between organic and mineral components, resulting in a protection against microbial degradation. Martin (1991) found a decrease in SOM decomposition in the long term when earthworms were present, possibly due to the physical protection of SOM in water-stable aggregates. Earthworms may also have a profound effect on soil aggregation and structure at the microaggregate scale. Several studies have indicated that during gut transit of the soil, the old microstructure is completely destroyed, but new microaggregates are formed within the casts (Shipitalo and Protz, 1988; Barois et al., 1993; Jongmans et al., 2001). In an incubation study, Bossuyt et al. (2004) confirmed that new microaggregates are very rapidly formed (!20 d (days)) within newly-excreted casts. However, the C protective capacity of these newly formed microaggregates was not investigated. Our objective was to investigate the effects of earthworms on (i) soil macro- and microaggregate formation, and (ii) protection of C at a microaggregate scale inside of their casts. We used 13C-labeled sorghum leaves, to follow the incorporation of newly-added residue into different aggregate-associated C fractions. Carbon mineralization rates of formed intact aggregates and crushed aggregates were determined to assess the amount of C protection from decomposition exerted by the different aggregate size classes.

2. Materials and methods 2.1. Site description and soils Surface (0–10 cm) no-tillage soil samples were collected with a shovel from the long-term agricultural experimental site (Horseshoe Bend) near Athens, GA, which is located in the Piedmont of the southern Appalachian Mountains (33854 0 N, 83824 0 W). The soil is a well-drained sandy clay loam (66% sand, 13% silt, 21% clay) in the Hiwassee series (fine kaolinitic thermic typic Kanhapludult). The area receives a mean annual precipitation of 1270 mm. The experimental plots (0.1 ha) were established in 1978 with replicated tillage treatments assigned in a complete randomized design. Details on the treatment histories at the Horseshoe Bend

Research Area Site can be found in Beare et al. (1994) and Hendrix (1997). After collection, the soil was air-dried (moisture content after air-drying 1–2%) and forced through a 250 mm sieve. The 250–1000 mm sized sand plus particulate organic matter fractions were kept and re-mixed with the soil after sieving. Stones larger than 1000 mm were discarded. 2.2. Formation of aggregates and earthworm casts This first experiment was designed to measure the effects of earthworms on the formation and distribution of soil aggregates. The incubation was conducted as described by Bossuyt et al. (2004). Briefly, the 250 mm sieved soil was subjected to three treatments, each with four replicates (nZ4): (i) no plant material and no earthworms; (ii) 13C labeled plant material, but no earthworms; and (iii) 13C labeled plant material and six adult earthworms [Aporrectodea caliginosa (Savigny 1826)]. Carbon-13 labeled sorghum (Sorghum bicolor (L.) Moench) leaves were used as the labeled plant material. All treatments consisted of 150 g soil. In the last two treatments, 1.2 g of plant material was mixed in with the soil; the mixture was brought to field capacity (11% water content) and put in glass jars. Tests showed that maximum aggregation occurred after 20 d. Therefore, samples were incubated at 20 8C for 20 d. Earthworms were added after 8 d. Respiration (total CO2 and 13CO2) was measured every day for 7 d and every other day afterwards for up to 20 d. At d 20, the earthworms were taken out of the jars and the soil was air-dried for 3 d to allow earthworm casts to stabilize (Marinissen and Dexter, 1990). It could be seen that the earthworms had processed a large amount of the soil in the jars. Aggregate size distribution was determined by wet sieving the capillary rewetted soil (Elliot, 1986). A series of three sieves was used to obtain four aggregate size fractions: (i) O2000 mm (large macroaggregates); (ii) 250–2000 mm (small macroaggregates); (iii) 53–250 mm (microaggregates); (iv) !53 mm (silt and clay fraction). Following wet sieving, the aggregate size fractions O53 mm were dried on the sieves in a dehumidifying chamber (10 8C). Particles !53 mm were collected in a bucket, total volume was measured and stirred and a subsample of a known volume was taken for analysis. 2.3. Protection of C inside newly formed aggregates and earthworm casts This second experiment was made to determine the effects of earthworm activity on the protection of C within earthworm casts by biologically determining the protected C and 13C pools inside the newly-formed casts and macroand microaggregates. Eight different sets of incubations were conducted: (1) and (2) intact large and small

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macroaggregates, (3) and (4) large and small macroaggregates crushed to !250 mm, (5) and (6) large and small macroaggregates crushed to !53 mm; (7) microaggregates and, (8) microaggregates crushed to !53 mm. Aggregates were crushed until !250 mm by gentle pressure with mortar and pestle and until !53 mm by grinding in a grinder (Spex 8000 Mixer/Mill, Spex Industries, Inc., Edison, NJ, USA). For all eight intact and crushed aggregate fractions from the first experiment (see Section 2.2), dry subsamples (12–15 g) were weighed into plastic cups, and deionized water was added to achieve field capacity. These subsamples were incubated (30 8C) in sealed jars with lids containing septa for gas sampling. Samples were incubated for 21 d since C and N mineralization assays are most often done for 21 d. Gas samples were taken on d 3, 11, and 21. 2.4. Analyses Total CO2 evolved during the incubations was analyzed on a Varian Star 3600CX (Varian Analytical Instruments, Sugar Land, Texas) gas chromatograph, which determined concentration based on thermal conductivity. Variations in 13 C of the CO2 evolved during the incubations were determined using a micromass VG optima mass spectrometer (Micromass UK Ltd., Manchester, UK). Results are expressed as: 13

C‰ Z ½ð13 Rsample =13 Rstandard Þ K 1 !1000

where 13 RZ 13 C=12 C and the standard is the international Pee Dee Belemnite (PDB). The amount of CO2–C derived from the sorghum residue (Qp) was calculated using the following mass balance:

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(4) and (5) protected C pools in large and small macroaggregates, (6) protected C pool in microaggregates and; (7) and (8) protected C pools in microaggregates within large and small macroaggregates. These different pools were calculated as follows (see also Fig. 1): (1) and (2) Unprotected C pools in large and small macroaggregatesZintact macroaggregate Cmin (3) Unprotected C pool in microaggregatesZintact microaggregate Cmin (4) and (5) Protected C pools in large and small macroaggregatesZ!250 mm crushed macroaggregate CminKintact macroaggregate Cmin (6) Protected C pool in microaggregatesZ!53 mm crushed microaggregate CminKintact microaggregate Cmin (7) and (8) Protected C pools in microaggregates within large and macroaggregatesZ!53 mm crushed macroaggregate CminKmacroaggregate-protected CKUnprotected macroaggregate C [or, replacing macroaggregate-protected C (see Eq. (4) and (5)) Z!53 mm crushed macroaggregate CminK(!250 mm crushed macroaggregate CminKintact macroaggregate Cmin)Kintact macroaggregate Cmin Z!53 mm crushed macroaggregate CminK!250 mm crushed macroaggregate Cmin with Cmin the cumulative C mineralized after 21 d of incubation. 2.6. Statistical analysis

Qt !dt Z Qp !dp C Qs !ds C Qb !db where Qt, the total amount of CO2–C; dt, its isotopic composition; Qp, the amount of CO2–C derived from the sorghum; dp, its isotopic composition (296G2.7‰); Qs, the amount of CO2–C derived from the soil; dsZits isotopic composition (K24.76G0.18‰); Q b, blank CO 2–C amount; db, its isotopic composition (K7.5G0.59‰). The control samples (no sorghum added) were used to measure Qs and ds, with the assumption of no priming effect. Total C and 13C from the aggregate size fractions was determined using a Finnigan Delta C Mass Spectrometer coupled to a Carlo Erba, NA 1500, CHN Combustion Analyzer via Finnigan’s Conflo II Interface. 2.5. Calculations The results of the aggregate incubations were used to define eight C pools in aggregates (Bossuyt et al., 2002): (1) and (2) unprotected C pools in large and small macroaggregates, (3) unprotected C pool in microaggregates,

The data were analyzed, using the SAS statistical package for analysis of variance (ANOVA-PROC GLM, SAS Institute, 1990). Separation of means was tested with the PDIFF option of the LSMEANS statement (means separation option: TUKEY) with a significance level of P!0.05.

3. Results 3.1. Formation of aggregates and earthworm casts 3.1.1. Water-stable aggregates There were significant effects of earthworm activity on the distribution of water-stable aggregates (Fig. 1). Large macroaggregates (O2000 mm) made up the largest proportion (w37% on average) of the whole soil for samples with earthworms and the proportion of large macroaggregates was on average 3.6 times greater than samples without earthworms. When no earthworms and no residue was added, no large macroaggregates were formed. The smaller

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Fig. 1. Visualization of unprotected and protected C pools in macroaggregates and microaggregates.

aggregate size classes (250–2000, 53–250 and !53 mm) made up a greater proportion of the soil without earthworms or residues. 3.1.2. Respiration There was no significant difference in total or residuederived cumulative respiration between samples with or without earthworms. Total respiration was 6.33 mg gK1 soil and residue-derived respiration was 4.41 mgKgK1 soil. The residue-derived respiration accounted for approximately 70% of the total respiration. 3.1.3. Total C and 13C concentrations Total C and 13C were significantly influenced by earthworm activity. Total C and 13C in large macroaggregates were significantly higher in the presence of earthworms (Figs. 2 and 3). There was no significant difference in total C or 13C in small macroaggregates. In the other aggregate size classes, total C and 13C were higher in the absence of earthworms. There were no significant differences in total C between the control samples and the samples without earthworms, except for the silt and clay fraction (!53 mm) in which total C was significantly higher when no residue was added.

3.2. Protection of C inside newly formed aggregates and earthworm casts 3.2.1. Unprotected and protected C and 13C pools in aggregates The amount of C mineralized after 21 d is shown in Table 1 as cumulative respired C (mg C kgK1 soil).

Fig. 2. Effects of earthworm activity on aggregate size distribution. Values followed by a different lowercase letter within aggregate size class are significantly different between sample treatments.

H. Bossuyt et al. / Soil Biology & Biochemistry 37 (2005) 251–258

Fig. 3. Effects of earthworm activity on total aggregate-associated C concentrations. Values followed by a different lowercase letter within aggregate size class are significantly different between sample treatments.

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macroaggregates was not detectable in the samples without earthworms because few large macroaggregates were present. The amount of 13C or residue-derived C mineralized after 21 d is shown in Table 1 as cumulative respired 13C (mg 13C kgK1 soil). The unprotected 13C pool in small macroaggregates was higher in the absence of earthworms. There was no difference between the treatments for the unprotected 13C pool in microaggregates. There was no protected 13C found in the large macroaggregates, small macroaggregates or microaggregates-within-small-macroaggregates. The protected 13C pool in microaggregateswithin-large-macroaggregates was a substantial pool in the presence of earthworms. Of the C in microaggregateswithin-large-macroaggregates, 22% was newly added residue C. Earthworm activity had no effect on the protected 13 C pool in microaggregates.

4. Discussion The unprotected C pools were always significantly larger than the corresponding protected C pools. The unprotected C pool in small macroaggregates was highest in the absence of earthworms and lowest in the absence of both earthworms and residue. The unprotected C pool in microaggregates was significantly higher in samples without earthworms than in those with earthworms. In the control soil, this pool was not significantly different from the other treatments. The protected C pools in small macroaggregates, microaggregates and microaggregates within small macroaggregates did not differ between treatments. The protected C pool in microaggregates within large macroaggregates was a large pool in the presence of earthworms and this pool was about 2.5 times higher than the protected C pool in the microaggregates within small macroaggregates. In contrast, the protected C pool in the microaggregates within large

4.1. Water-stable aggregates The presence of the endogeic earthworm A. caliginosa, had significant effects on the formation of large macroaggregates. In the samples where earthworms were added, higher amounts of water-stable large macroaggregates (O2000 mm) were found. Several researchers have described the positive influence of earthworms on the formation and stability of soil aggregates. Martin and Marinissen (1993) described how earthworms play an important role in the production of binding agents responsible for the formation of water-stable macroaggregates. van Rhee (1977), De Vleesschauwer and Lal (1981) and McKenzie and Dexter (1987) showed a higher stability in earthworm casts than in the surrounding soil aggregates. Earthworms ingest large quantities of organic materials that are mixed and excreted as casts (Parmelee et al., 1990;

Table 1 C and 13C pools associated with aggregates in soil samples with or without earthworms

(1)

(2)

(3)

(4) (5)

Large macroaggregates (O2000 mm) Unprotected C Protected C Small macroaggregates (250–2000 mm) Unprotected C Protected C Microaggregates (53–250 mm) Unprotected C Protected C Microaggregate within large macroaggregates Protected C Microaggregate within small macroaggregates Protected C

Total carbon mineralized (mg kgK1 soil)

13

CWorms

KWorms

KResidue

CWorms

KWorms

260.8 6.0

n.d* n.d

0 0

177 0.0

n.d n.d

179.5b 1.2a

283.7a 0.0a

129.8c 10.7a

137b 1.1a

206a 0.0a

168.8b 55.8a

260.9a 72.4a

238.4ab 70.8a

63.3a 8.8a

72.9a 6.3a

161.7

n.d

0

35.2

n.d

62.4a

42.5a

44.1a

0.0a

0.0a

Carbon mineralized (mg kgK1 soil)

Values followed by a different lowercase letter across the table are significantly different between sample treatments. *n.d, not detectable.

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Martin and Marinissen, 1993; Je´gou et al., 1998) and improve stable macroaggregation (Guggenberger et al., 1996; Marinissen and Hillenaar, 1996; Scullion and Malik, 2000). 4.2. Respiration There was no effect of earthworm activity on overall soil respiration or residue-derived respiration. Earthworms are known to have significant effects on microbial activity providing labile C substrates and favorable conditions in the gut and casts. However, the contribution of the earthworms themselves to total respiration is usually low. Peterson and Luxton (1982) reported that the soil fauna generally appears to be responsible for less than 5% of total decomposer respiration. Satchell (1971), Persson and Lohm (1977) and Lamotte (1989) also found that micro-organisms are the major contributors to soil respiration being responsible for 80–95% of the total CO2 respired. Zhang and Hendrix (1995), on the other hand, found an increase in microcosm respiration up to w22% when earthworms were present compared to control soil. Haimi and Huhta (1990) observed that 15–18% of microbial respiration was due to earthworm activity in raw humus forest soil. 4.3. Total C and

13

C concentrations

Our results showed that earthworms induced a redistribution of total C and residue-derived C into the large macroaggregates or the earthworm casts. Total and residue-derived aggregate-associated C concentrations in large macroaggregates were, respectively 3- and 2-fold in the presence of earthworms. Zhang and Schrader (1993) found an increase of 20–37% of total C in casts compared to bulk soil and Lee (1985) concluded that C content of casts is usually about 1.5–2-fold than the surrounding soil. Shipitalo and Protz (1988), Daniel and Anderson (1992), Barois et al. (1993) and Buck et al. (1999) reported a larger amount of total or organic C in earthworm casts compared to bulk soil. Selective feeding on particles high in organic matter may result in larger amounts of C in the earthworm casts. For example, Bhandari et al. (1967) suggested that the increase in microbial activity and an increase in polysaccharide production in casts caused a higher OM concentration compared to bulk soil.

due to physical protection within aggregates and that the other part is due to the grinding of the plant material. Bossuyt et al. (2002), however, reviewed literature on the effects of grinding on plant residue mineralization and concluded that the grinding of organic particles is probably not an important factor contributing to increased C mineralization. Therefore, our methodology to assess physical protection exerted by aggregates seems to be valid. The largest pools were unprotected pools. This suggests that after 20 d of incubation, most of the aggregateassociated C was still in an unprotected form and easily mineralizable by the microbial community. Crushing the macroaggregates to the size of microaggregates made macroaggregate-protected C and 13C available for microbial mineralization. These pools were very small or non-existent after 20 d of incubation, showing that, in the short term, almost no C or 13C was protected at a macroaggregate scale in both the samples with or without earthworms or the control soil (Fig. 4). When aggregates were ground to !53 mm, however, a significant amount of C was mineralized from the large macroaggregates in the presence of earthworms. This showed that C protection due to microaggregates within macroaggregates is much more important than C protection in macroaggregates per se and that earthworms play a very important role in protecting C inside microaggregates within macroaggregates. This pool was not measurable and therefore insignificant in the absence of earthworms because the amount of large macroaggregates was only one quarter of the amount in the earthworm treatment. In current aggregate-SOM concepts (e.g. Golchin et al., 1994; Six et al., 1998; Gale et al., 2000), the formation of stable microaggregates within macroaggregates and the concomitant protection of C is understood as a longer-term

4.4. Unprotected and aggregate-protected C and 13C pools When aggregates are ground, C that was previously physically protected within the aggregates becomes available to microbial activity. But since the grinding of aggregates potentially breaks up the plant material present within the aggregates, and since it has been suggested that C mineralization increases with decreasing residue particle size, it is possible that only part of the C mineralized is

Fig. 4. Effects of earthworm activity on aggregate-associated 13C concentrations. Values followed by a different lowercase letter within aggregate size class are significantly different between sample treatments.

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aging and stabilization process of organo-mineral complexes mediated by microbial activity and exudation. However, Bossuyt et al. (2004) showed that earthworms induce a fast formation of these microaggregates and here we show that these microaggregates do protect a significant amount of C after 12 d of incubation. Of the total C protected inside microaggregates within large macroaggregates, 22% was fresh residue-derived C, showing that a significant amount of freshly added residue is already incorporated and protected inside microaggregates within macroaggregates. Earthworms incorporate fresh organic residues directly into the soil matrix and microbial decomposition is not necessarily required for the formation of microaggregates (Pulleman et al., 2002). These microaggregates are formed within earthworm casts before the casts are even excreted (Shipitalo and Protz, 1989; Barois et al., 1993). 4.5. Conclusions We can conclude, as others have, that earthworms significantly affect the formation of soil aggregates and their associated C pools. The proportion of large macroaggregates is significantly higher in the presence of earthworms and such aggregates contain significantly more total C and residue-derived C compared to bulk soil. Earthworms were found to form a significant pool of protected C in microaggregates within large macroaggregates after 12 d of incubation. This rapid protection of C in microaggregates within large macroaggregates is much greater than the protection in either large of small macroaggregates. While our study clearly shows the effects of earthworm activity on C protection in casts, we realize it was a short-term laboratory incubation. Further field studies, where interactions with plants, wetting/drying and freezing/thawing cycles and with other earthworm species, are needed.

Acknowledgements Thanks to Dana Camp for laboratory assistance, to Dr Miguel Cabrera for use of a gas chromatograph and to Tom Maddox for C analyses. This research was supported by grants from the National Science Foundation.

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