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How do earthworms influence organic matter quantity and quality in tropical soils?
Soil Biology & Biochemistry 43 (2011) 223e230
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How do earthworms influence organic matter quantity and quality in tropical soils? Hanh Nguyen Hong a, b, Cornelia Rumpel c, Thierry Henry des Tureaux a, b, Gérard Bardoux c, Daniel Billou c, Toan Tran Duc a, Pascal Jouquet a, b, * a b c
IRD e IWMI e SFRI, Dong Ngac, Tu Liem, Hanoï, Viet Nam IRD, UMR 211 BIOEMCO, Centre IRD Ile de France, Bondy, France CNRS, UMR 7618 BIOEMCO, Campus Paris AgroTech, Thiverval-Grignon, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 June 2010 Received in revised form 22 September 2010 Accepted 24 September 2010 Available online 9 November 2010
Earthworms are important regulators of soil structure and soil organic matter (SOM) dynamics; however, quantifying their influence on SOM cycling in tropical ecosystems remains little studied. Simulated rainfall was used to disrupt casts produced by Amynthas khami and their surrounding soil (control) into a range of small sized aggregates (50e250, 250e500, 500e2000 and 2000e5000 mm). To gain insight into how earthworms influence SOM biogeochemical composition in the aggregates, we carried out elemental and stable isotope analysis, and analytical pyrolysis (Py GC/MS). We also characterized their lignin component after oxidation with cupric oxide (CuO). The C content of smaller size fractions (<500 mm) in the control soil was higher than in the larger fractions. Our study therefore suggests that the aggregate hierarchy concept, which is used to understand soil aggregates and SOM dynamics in temperate soils, may not be applicable to the tropical Acrisol studied here. Earthworms modified SOM organization in soil aggregates. Although the isotope analyses were useful for highlighting SOM enrichment in the earthworm casts, aggregate fractions could not be classified according to particle size. Molecular analyses were necessary to indicate that SOM in all size fractions of casts consisted of relatively undecomposed material. Protection of the most labile SOM structures occurred in the smallest aggregate size fraction (50e250 mm). Py GC/MS showed that earthworm casts and control aggregates <2000 mm could be clearly distinguished according to the molecular properties of their SOM. Aggregates larger than 2000 mm, however, were most probably composed of all fractions and were not different. As a consequence, our results indicate that studies to determine the impact of earthworms on SOM turnover in soil are spatially dependant on the scale of observation. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Earthworms SOM Soil aggregate organization Pyrolysis GC/MS Lignin
1. Introduction Soil organic matter (SOM) is an important active carbon reservoir at the global scale and plays a pervasive role in promoting soil ecological functions. By contributing greatly to a number of soil properties including soil structure, porosity, water retention, cationic exchange and pH buffering capacities, SOM is a fundamental component, ensuring soil fertility (Lal, 2004; Weil and Magdoff, 2004). Understanding the dynamics of SOM is therefore of primary importance and requires knowledge of the factors affecting its stability. Soil aggregates have been proposed as
* Corresponding author. IRD, UMR 211 BIOEMCO, Centre IRD Ile de France, Bondy, France. E-mail address: [email protected] (P. Jouquet). 0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.09.033
structural units within the soil that control the dynamics of SOM and nutrient cycling (e.g., Tisdall and Oades, 1982; Chevallier et al., 2004). They are usually considered as organized units in the form of micro- (50e250 mm) and macroaggregates (>250 mm) occluding particulate organic matter (POM) (see Six et al., 2004 for a review). Several studies focused on the mechanisms by which earthworms influence SOM sequestration within soil aggregates (See a review in Lavelle and Spain, 2001). Soil aggregates are completely reorganized after their passage through earthworm guts (Shipitalo and Protz, 1989; Barois et al., 1993). SOM is partially digested by earthworms and one proportion becomes physically protected in microaggregates (i.e., Bossuyt et al., 2004, 2005; Fonte et al., 2007). This physical protection is explained by the higher water stability of casts compared to the surrounding soil, because: (i) microbial polysaccharides and other organic products strengthen bonds between organic and mineral components (Shipitalo and Protz,
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1989); (ii) casts have higher bulk density and/or the presence of a cortex on their periphery which renders them impermeable to water (Blanchart et al., 1993, 1999; Jouquet et al., 2008a). Most of the studies dealing with the influence of earthworms on soil properties have focused on the total C and N content, and the subsequent C:N ratio of SOM as an indicator of soil quality (i.e., Desjardins et al., 2003; Coq et al., 2007; Eisenhauer et al., 2007; Fonte et al., 2007). More recently, stable isotopes were also used to estimate the trophic position of earthworms or the origin of SOM in their casts (i.e., Bossuyt et al., 2004, 2005; Fonte et al., 2007; Don et al., 2008; Hyodo et al., 2008; Pollierer et al., 2009). Although measuring total C and N content might be interesting for estimating the impact of earthworms on the C budget at the ecosystem scale, it can also been considered as non-exhaustive and does not contribute to a clear understanding of how earthworms influence SOM mineralization and sequestration. Molecular based information on OM chemistry may provide additional information on this subject (Guggenberger et al., 1996). In this study we focused on “giant” earthworm casts that are very commonly produced in the tropics (Fig. 1). Our objectives were (i) to assess the relevance of the stoichiometric approach to describe SOM quality through its C and N content, and (ii) to evaluate what information can be gained from a biogeochemical analysis of aggregate size fractions concerning the effect of earthworm activity on SOM composition and its protection.
2. Material and methods 2.1. Study site and study model Soil and cast aggregates were sampled in the experimental catchment (46 ha) of the MSEC (Management Soil Erosion Consortium of the International Water Management Institute, IWMI) project (Valentin et al., 2008). This study site is located in Dong Cao village, in north-eastern Vietnam, approximately 50 km south-west of Hanoi (20 570 N, 105 290 E). The annual rainfall ranges from 1500 to 1800 mm, of which 80e85% occurs from April to October. The air humidity is always high, between 75 and 100%. The mean daily temperature varies from 15 C to 25 C. The soil is an Acrisol (WRB, 2006) with more than 50% clay, mainly kaolinite, with a low pH of around 5, and a low CEC (<10 cmol kg soil1) (Jouquet et al., 2008a,b; Podwojewski et al., 2008). Earthworm casts were produced by Amynthas khami. A. khami varies in size and adults can reach up to more than 50 cm in length. It builds casts that are partly deposited on the soil surface (Fig. 1) but does not influence soil particle size (around 66% clay, 27.5% silt and 6.4% sand, Jouquet et al., 2007). Casts are globular and characterized by a very high soil structural stability (Jouquet et al., 2008a). The abundance of A. khami in the experimental watershed was previously reported by Jouquet et al. (2008b) and reaches 1.11 (0.59) ind m2. 2.2. Soil sampling
Fig. 1. Casts produced by Amynthas khami. Fecal aggregates are deposited one on top of the other [photo, P. Jouquet, 2009].
Soil and cast aggregates were collected in a fallow field following a Eucalyptus plantation which was cut down in 2003. In this area, there was no soil preparation and no herbicides were applied but the vegetation cover was maintained at a low level (<50 cm) by livestock grazing. Casts (CAST) were collected on the soil surface and control soil aggregates (CONTROL) were sampled in three different randomly chosen areas in the fallow. The number of repetitions was therefore n ¼ 3. CAST and CONTROL constitute the two dominant free soil surface aggregates which are fragmented by raindrop impacts at the beginning of the rainy season (Jouquet et al., 2010). For that, samples were taken few days before the rainy season, in March 2008. CAST were collected on the soil surface. They were carefully selected according to their aspect and only rounded shape aggregates without trace of alteration by the rain were sampled. A previous study showed that CAST lose their rounded shape aspect very rapidly and get an angular aspect when affected by the rain (Jouquet et al., 2009). We assumed that this sampling design allowed us to sample dry CAST which had approximately the same history (i.e., they had been produced several months before, probably at the end of the previous rainy season in October 2007). Microbial activity is higher in freshly emitted earthworm casts and then decreases with time (Lavelle and Spain, 2001). Sampling soil aggregates at the end of the dry season after several months of incubation therefore also allowed us to reduce the variability which is inherent at any soil sampling. In March 2008, tree seedlings were planted in the fallow and many dry soil clods (>5000 mm) formed during soil preparation (no tillage but shallow holes with a spade) were deposited on the soil surface. These soil aggregates were carefully sampled and inspected to be sure that there was no trace of earthworm activity (casts or galleries) and were considered as CONTROL. Soil samples were air-dried for three days. In addition, a large amount fresh leaves were randomly sampled from different non-identified plant species in each plot within the studied area. The samples were airdried for three days and thoroughly mixed to produce three composite samples (one per plot). A similar procedure was applied for root sampling.
H.N. Hong et al. / Soil Biology & Biochemistry 43 (2011) 223e230
2.3. Rainfall simulation Simulated rainfall was used to fragment soil aggregates. The rainfall intensity was 44 mm h1 (i.e., kinetic energy w268 J m2), which was estimated to be equivalent to a medium intensity rainfall event. About 80 g of CAST aggregates, corresponding to approximately one individual earthworm cast, or 80 g of CONTROL aggregates were put on a 500 mm sized mesh grid (15 15 cm) which covered a 10 L bucket in order to recover water and aggregates that passed through the grid. Because CAST are more stable than CONTROL aggregates (Jouquet et al., 2008a), aggregates which remained on the grid were collected after 1 and 2 h of rainfall simulation, respectively for CONTROL and CAST aggregates. Soil aggregates remaining on the grid were gently sieved in distilled water and carefully separated into 4 size classes: 50e250, 250e500, 500e2000 and 2000e5000 mm. These different fractions were air-dried for 2 days. All the procedures were repeated for each sampled area (n ¼ 3). 2.4. Elemental and stable isotope analyses The organic carbon (C) and nitrogen (N) content of each aggregate size class were determined by the dry combustion method using a CHN elemental analyzer (CHN NA 1500, Carlo Erba). Analytical precision was 0.1 mg g1 for C and 0.05 mg g1 for N content. The C and N isotope ratios of aggregate size classes of CAST and CONTROL aggregates were compared to those of roots and leaves. Stable isotope ratios were determined with an elemental analyzer (EA, Eurovector) coupled to an isotope ratio mass spectrometer in continuous flow mode (Isoprime GV instruments). C and N isotope ratios were defined using the delta notation:
dsample (&) ¼ [(Rsample Rstandard)/Rstandard] 1000 where R is the isotopic ratio 13C/12C and 15N/14N for the sample and reference gas, Rsample and Rstandard, respectively. The stable isotope ratios were calculated relative to the Pee Dee Belemnite standard (PDB) for C and relative to atmospheric N2 for N. Analytical precision for isotope measurements was 0.1& for C and 0.2& for N isotopes. 2.5. Mineral-bound OM We assessed the amount of mineral-bound OM, which is usually protected against microbial attack (Baldock and Skjemstad, 2000) after mineralization of the sample using hydrofluoric acid (HF). This treatment was found to isolate mineral-bound carbon without affecting the OM composition of the remaining material (Rumpel et al., 2006). The HF treatment was performed on aggregate size fractions by adding 10 ml of 10% HF to 2 g ground mineral soil material (Eusterhues et al., 2007). The suspensions were shaken for 2 h at room temperature, then centrifuged and the supernatant removed. This procedure was repeated five times. Then the residues were washed five times with deionised H2O to remove salts and residual HF. Afterwards the samples were freeze-dried and weighed. The concentration of organic carbon (OC) was determined and the amount of OC lost upon this treatment was calculated by mass balance calculation. 2.6. Lignin content and composition The lignin content and composition of the samples were determined through release of the phenol monomers by alkaline CuO oxidation (Hedges and Ertel, 1982). This method allows both
225
parameters to be assessed quantitatively, which gives an indication of (i) the presence of intact plant material and (ii) the state of OM decomposition. Briefly, 50 mg of pure organic matter and 500 mg of the mineral A horizon were oxidized with 250 mg CuO and 2 M NaOH at 172 C under N2 for 2 h. After purification using solidphase extraction with a C18 column (International Sorbent Technology) as suggested by Kögel and Bochter (1985), lignin phenols were derivatised and quantified as trimethylsilyl derivatives by gas chromatography. We used a HP GC 6890 gas chromatograph equipped with a flame ionisation detector. Separation of the individual monomers was achieved with a SGE BPX-5 column (50 m length, 0.25 mm inner diameter, 0.32 mm coating). The temperature was programmed from 100 C (1 min isotherm) to 150 C at 3 C min1 and from 150 to 300 C at 1 C min1. The detector temperature was kept at 350 C. Helium was used as carrier gas (1 ml min1). This method yields a suite of phenolic CuO oxidation products, such as vanillyl (V), syringyl (S), and cinnamyl (C) compounds with aldehydic, ketonic, and acidic side chains. The sum of CuO oxidation products (VSC) is usually taken as an indicator of the total amount of lignin and ratios between the acidic and the aldehydic forms of the vanillyl and syringyl compounds (Ac/ Al)V,S indicate their degree of decomposition (Hedges et al., 1988). VSC is decreasing and the (Ac/Al)V,S are usually increasing with increasing OM degradation. C- and S-units are less stable than V units and therefore decreasing C/V as well as S/V ratios indicate more advanced stages of OM decay (Bahri et al., 2006). Analytical precision was 10% for VSC and 5% for ratios between phenolic products. 2.7. Analytical pyrolysis Analytical pyrolysis consists of curie-point pyrolysis of OM and identification of pyrolysis products using gas chromatography coupled to mass spectrometry (Py GC/MS). It gives a good overview of the molecular composition of the bulk material in terms of the contribution of polysaccharides, N-containing, lignin-derived and aliphatic compounds. Curie-point pyrolysis was carried out with a pyrolysis unit (GSG Curie-point Pyrolyser 1040 PSC) coupled to a gas chromatograph (Hewlett Packard HP 5890) and a mass spectrometer (Hewlett Packard HP 5889; electron energy 70 eV). Approximately 0.5e1 mg of HF-treated sample were loaded in tubular ferromagnetic wires and inductively heated to their Curie temperature at 650 C during 0.15 s in the pyrolysis unit. The pyrolysis products were transferred to the GC system using the splitless mode with He as the carrier gas. The pyrolysis products were separated on a 60 m fused silica capillary column with a wax stationary phase (SolGelWax, SGE, 0.32 mm i.d., film thickness 0.5 mm). The temperature of the GC oven was programmed from 30 C to 280 C at 2 C min1. The final temperature was held for 15 min. We chose a polar column, because we were interested in the relative quantification of polysaccharide-derived and ligninderived compounds as well as N-containing compounds from the GC traces. These compounds are most likely constituting the bulk of SOM in tropical clayey soils (Rumpel et al., 2006). Such compounds were found to yield broad peaks when separated on a non-polar column (Dignac et al., 2005). We identified five types of pyrolysis products according to their origin: (1) polysaccharide-derived compounds; (2) lignin-derived compounds; (3) aliphatic compounds derived from lipids or other aliphatic biomacromolecules; (4) N-containing compounds most likely derived from proteins or other N-containing molecules and (5) non-specific compounds derived from more than one of the other substance classes. The peak areas of the Total Ion Current (TIC) trace were integrated using the GC ChemStation program
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(Agilent Technologies). The total area of the five types of pyrolysis products was calculated as the sum of the identified peaks and the relative contribution of each compound class to this sum was reported.
Table 1 Repeated measures ANOVA table for the effect of soil aggregate origin (CAST vs. CONTROL) and size (50e250, 250e500, 500e2000 and 2000e5000 mm) on the C and N content and C and N isotope ratios. F-values are displayed. ***p < 0.001, **p < 0.01, *p < 0.05, ns p > 0.05. C
2.8. Statistical analyses Before analyses, data were tested for homogeneity of variance using the Levene’s test. Data were analyzed by repeated measures analysis of variance (ANOVA) with the origin of the soil aggregates (CAST vs. CONTROL) and the soil aggregate sizes (50e250, 250e500, 500e2000 and 2000e5000 mm) as the independent variables and the aggregate origin as within-subject factor. Comparisons between means were tested with LSD test. A Principal Component Analysis (PCA) was carried out to differentiate the samples based on their pyrolysis signature. Briefly, we used a matrix of 24 samples and the relative contribution of the five types of pyrolysis products previously described. All statistical calculations were carried out using R (R Development Core Team, 2008). Differences among treatments were declared at the p < 0.05 probability level of significance. 3. Results 3.1. SOM content and stable isotope composition The organic C and N content in soil aggregates are shown in Fig. 2 and results of the statistical analyzes are shown in Table 1. CAST aggregates were characterized by a higher C and N content than CONTROL aggregates for all the aggregate size classes. C and N contents were not influenced by aggregate size for CAST.
Origin (1) Aggregate size (2) (1) (2)
N
d13C
d15N
df
F
F
F
F
1 3 3
123.8*** 5.3* 2.7 ns
109.5*** 3.6* 2.9 ns
95.0*** 17.1*** 15.1***
55.7*** 1.8 ns 7.2**
Conversely, CONTROL soil aggregates smaller than 500 mm had significantly more C and N than those bigger than 500 mm. Fig. 3 shows the d13C and d15N in CAST, CONTROL and in the composite samples of roots and leaves. Stable isotope ratios were dependent on soil origin and aggregate sizes (Table 1). Interestingly, the smaller size fractions were depleted in 13C and 15N compared to the bigger size fractions. CAST samples were characterized by a signature closer to that of leaves and roots compared to CONTROL samples, which generally showed higher 13C and 15N enrichment. d13C in CASTaggregates showed increased 13C enrichment compared to leaves and 13C depletion compared to roots and CONTROL aggregates. The d15N signatures of both types of aggregates were enriched in 15N with regards to the plant material. CAST aggregates smaller than 250 mm had a significantly different d13C and d15N signatures compared to those larger than 250 mm. For CONTROL samples, although no significant differences were observed for aggregates between 50e250 mm and 250e500 mm, aggregates between 500e2000 mm and >2000 mm had specific d13C and d15N signatures. 3.2. Mineral-bound OM Demineralization of soil samples by treatment with hydrofluoric acid led to a significantly higher reduction in soil weight for CONTROL compared with CAST aggregates (93.5 vs. 90.6%, SE: 0.18 and 0.15, for CONTROL and CAST aggregates, respectively; Table 2), without a significant effect of aggregate size. As a consequence, more mineralbound C was lost for CONTROL samples than for CAST samples, whatever the aggregate sizes (Fig. 4). For both CAST and CONTROL, more important C losses were observed for aggregates larger than 2000 mm, and no difference were found between the other aggregate size classes. The same trend was observed with N loss.
Fig. 2. Total C and N content (%) in cast (CAST, in black) and control (CONTROL, in white) soil aggregates according to their sizes (>2000, 2000e500, 500e250, 250e50 mm). Histograms with the same letters are not significantly different at p ¼ 0.05. Bars represent the standard errors.
Fig. 3. d13C and d13N values for leaves and roots (triangle), casts (sphere) and control (square) soil aggregates of different size classes (>2000, 2000e500, 500e250, 250e50 mm). Data within the same ellipse are not significantly different at p ¼ 0.05. Bars represent the standard errors.
H.N. Hong et al. / Soil Biology & Biochemistry 43 (2011) 223e230 Table 2 Repeated measures ANOVA table for the effect of soil aggregate origin (CAST vs. CONTROL) and size (50e250, 250e500, 500e2000 and 2000e5000 mm) on the amount of soil weight and C and N loss after hydrofluoric acid treatment. F-values are displayed. ***p < 0.001, *p < 0.05, ns p > 0.05. Soil loss
Origin (1) Aggregate size (2) (1) (2)
C loss
N loss
df
F
F
F
1 3 3
35.3 *** 0.2 ns 1.4 ns
24.5*** 28.3*** 3.6*
7.4* 21.3*** 17.5***
3.3. Lignin analyses The total lignin contribution to SOM content, estimated by the sum of vanillyl (V), syringyl (S) and cinnamyl (C) units (VSC), was significantly higher in CAST than in CONTROL samples <2000 mm without a significant influence of aggregate size (Table 3). Conversely, CAST >2000 mm had similar VSC content than all the CONTROL aggregate size classes. CAST aggregates were characterized by a higher amount of C units compared to the CONTROL samples, whereas no difference was observed in the amount of V and S units. C units in CAST had significantly higher concentrations in microaggregates (50e250 mm) compared to bigger aggregates and their CONTROL counterparts. The acid to aldehyde ratio of vanillyl (Ac/Al)v was higher in CONTROL samples >2000 mm than in CAST of the same size, without any significant difference for the other aggregate size fractions. This is in line with the elevated C/V and S/V ratios in CAST >2000 mm compared to CONTROL >2000 mm and could indicate that lignin in the CONTROL samples was more degraded than in CAST samples. No significant differences were observed for (Ac/Al)s between CAST and CONTROL aggregates of the same size.
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3.4. Assessment of SOM quality by analytical pyrolysis (Py GC/MS) From the total ion chromatograms, a number of pyrolysis products were identified, which were derived from five different biochemical families. All pyrograms showed peaks related to polysaccharide-derived, lignin-derived, N-containing, aliphatic and non-specific compounds (data not shown) (Fig. 5). Pyrolysis products with a furan, cyclopentene and cyclopropane structure were identified as the main polysaccharide-derived compounds. Ligninderived compounds were dominated by methoxy phenols, which indicate the contribution of guaiacyl lignins (Nierop et al., 2001). Consistent with the results obtained by CuO oxidation, the occurrence of pyrolysis products based on dimethoxy phenols indicates the presence of syringyl lignin. N-containing compounds include pyrrols and pyridines. Pyrrol and its derivatives are formed during the pyrolysis of proteins by cyclization (Chiavari and Galletti, 1992), but may also be pyrolysis products of chlorophyll (Bracewell et al., 1987). Additionally, many non-specific compounds were released during pyrolysis of the three soils. Acetic acid, phenol, methyl phenols, styrene and toluene are non-specific compounds, since they are derived from the pyrolysis of protein, polysaccharides, lignins peptides and tannins (Saiz-Jimenez and Leeuw, 1984). For CAST samples, a large number of lignin-derived pyrolysis products and fewer N-containing compounds contributed to the total pyrolysis products identified. Principal Component Analysis (PCA) of the relative contribution of the five types of pyrolysis products is shown in Fig. 6. Axis 1 and 2, respectively, explained 44.4 and 25.2% of the total data variance. Axis 1 is mostly determined by the contribution of N-containing and polysaccharide-derived compounds. The PCA plot of the samples in the plane shows that CAST and CONTROL aggregates <2000 mm from the same size class were clearly separated on the first and second axis according to their biogeochemical composition as seen by Py GC/MS. The molecular composition of CAST aggregates did not allow us to clearly distinguish between aggregates <2000 mm within the CAST and CONTROL samples. In addition, aggregates >2000 mm were characterized by a high variance and overlapped all other size fractions. 4. Discussion 4.1. Relevance of the soil aggregate hierarchy in Acrisols
Fig. 4. Total amount of C and N lost (%) after treatment of the samples (earthworm casts in black; control soil in white) with hydrofluoric acid. Histograms with the same letters are not significantly different at p ¼ 0.05. Bars represent the standard errors.
Soil aggregate organization is usually described by comparing micro- and macroaggregrate soil properties (see Six et al., 2004 for a review). Microaggregates (50e250 mm) are especially important in regulating SOM dynamics. They are characterized by a high structural stability, which results in a low rate of SOM turnover. Conversely, macroaggregates (250e2000 mm) are less stable and the SOM turnover rate increases (Six et al., 2000, 2002). However, aggregate hierarchy and the relevance of micro- and macroaggregate classification for understanding SOM dynamics have limitations in highly weathered tropical soils, such as Oxisols (Zoratelli et al., 2005, 2007). We are not aware of any studies that describe C and N pools in micro- and macroaggregates in Acrisols. In our study, the highest SOM content in small size aggregates (<500 mm) contradicts the concept of aggregate hierarchy presented by Tisdall and Oades (1982), who suggested that larger aggregates are more enriched in SOM than smaller aggregates. However, we did confirm that a distinction exists between small and larger size aggregates due to their SOM content, with an alternative functional limit observed at 500 mm and not 250 mm, as usually observed in temperate soils (Six et al., 2004). We assume that this might be explained by the ability of Acrisols to produce water-stable pseudosilts and pseudo-sand aggregates due to the high reactivity of
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Table 3 Lignin contribution to SOM content (mg gC1), estimated by the sum of vanillyl (V), syringyl (S) and cinnamyl (C) units (VSC) and ratios between the acidic and the aldehydic forms of the vanillyl and syringyl compounds (Ac/Al)V,S in CAST and CONTROL samples according to their sizes (>2000, 2000e500, 500e250, 250e50 mm). Standard errors in parenthesis, n ¼ 3. F-values of repeated measures ANOVA on the effect of soil aggregate “origin” (CAST or CONTROL) and “size” (50e250, 250e500, 500e2000 and >2000 mm) factors and their interactions. ***p < 0.001, **p < 0.01, *p < 0.05, ns p > 0.00.5. V
S
C
CAST >2000 mm 2000e500 mm 500e250 mm 250e50 mm
3.14 6.18 5.05 7.12
(0.37) (1.37) (1.25) (1.66)
2.78 4.59 3.35 4.57
(0.43) (0.93) (0.92) (1.05)
1.64 2.79 3.00 5.12
CONTROL >2000 mm 2000e500 mm 500e250 mm 250e50 mm
4.59 5.15 4.36 2.68
(1.70) (1.42) (0.52) (0.08)
1.89 3.82 2.23 1.74
(0.62) (1.32) (0.15) (0.01)
1.35 2.36 1.85 1.61
F-values Origin Size Origin*Size
0.8 ns 0.8 ns 2.1 ns
1.4 ns 2.0 ns 0.8 ns
VSC
S/V
(0.30) (0.41) (0.61) (1.04)
7.56 (0.25) 13.56 (2.53) 11.40 (2.78) 16.83 (3.70)
0.95 0.76 0.65 0.65
(0.27) (0.11) (0.04) (0.04)
0.52 0.47 0.61 0.73
(0.04) (0.04) (0.03) (0.04)
0.65 0.64 0.93 1.13
(0.48) (0.79) (0.09) (0.06)
7.83 (2.79) 11.33 (3.41) 8.44 (0.62) 6.03 (0.05)
0.42 0.76 0.52 0.65
(0.03) (0.13) (0.04) (0.02)
0.29 0.46 0.44 0.60
(0.01) (0.06) (0.06) (0.04)
0.71 0.62 0.84 0.92
1.6 ns 1.5 ns 2.4 ns
0.4 ns 0.6 ns 2.0 ns
2.8 ns 3.8* 3.6*
oxides, and/or the method we used to produce the aggregate size classes (rainfall simulation plus wet sieving in our study vs. wet sieving alone in the other studies). Acrisols are tropical and subtropical soils, which are less weathered than Oxisols (i.e., Ferralsols; WRB, 2006). Further studies are therefore needed to confirm our observation, i.e., the evolution of micro-aggregate sizes in Acrisols, and to determine the limitations and magnitude of the aggregate hierarchy hypothesis. Conversely in casts, the C and N content did not vary according to the aggregate sizes, suggesting that those made by earthworms (CAST) had a homogeneous organization. This finding is in agreement with the fact that casts produced by A. khami are described as an accumulation of small size aggregates <500 mm into bigger size aggregates (>2000 mm), while the control surrounding soil is characterized by a hierarchical organization of smaller aggregates (<500 mm) inside larger water-stable aggregates (>500 mm) (Jouquet et al., 2009).
C/V
C/S
1.4 ns 12.4*** 2.0 ns
(Ac/al)v
(Ac/al)s
(0.20) (0.09) (0.07) (0.11)
0.31 0.86 0.47 0.56
(0.08) (0.21) (0.07) (0.11)
0.83 0.60 0.45 0.45
(0.30) (0.16) (0.04) (0.04)
(0.03) (0.04) (0.05) (0.03)
1.00 0.75 0.79 0.58
(0.17) (0.09) (0.09) (0.06)
0.76 0.57 0.53 0.54
(0.28) (0.06) (0.02) (0.01)
0.3 ns 6.5 ** 0.6 ns
0.1 ns 1.6 ns 4.9*
0.4 ns 4.2* 0.3 ns
A. khami, which involves consuming fresh organic debris on and/or in soil. The use of stable isotope techniques is a useful complementary method for exploring the feeding ecology of earthworm field populations (Neilson et al., 2000; Scheu et al., 2002; Curry and
4.2. SOM content and quality as an indicator of earthworm ecological niche Classically, earthworm casts are said to be enriched in SOM compared to their surrounding soil environment (Lavelle and Spain, 2001). Our study confirms this general trend and C and N contents were significantly higher in earthworm casts than the surrounding soil. This is because of the feeding behavior of
Fig. 5. Proportion of pyrolysis products identified in CAST and CONTROL samples. POLYSAC: polysaccharide-derived compounds; LIG: lignin-derived compounds; ALIPHATIC: aliphatic compounds; N: N-containing compounds; NON-SPECIFIC: nonspecific compounds.
Fig. 6. Principal components analysis (PCA) carried out with the organic matter quality measured by Pyrolise in casts (CAST) and control (CONTROL) soil aggregates according to their sizes (>2000, 2000e500, 500e250, 250e50 mm). (a) Eigenvalue diagram. (b) Correlation circle on F1eF2 plane. Variables are polysaccharide-derived compounds (POLYSAC), lignin-derived compounds (LIG), aliphatic compounds derived from lipids or other aliphatic biomacromolecules (ALIPHATIC), N-containing compounds most likely derived from proteins or other N-containing molecules (N) and unspecific compounds derived from more than one of the other substance classes (NONSPECIFIC). (c) Soil structure ordered in the plane defined by factors 1 and 2. Ellipses represent 90% confidence limits.
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Schmidt, 2007; Hyodo et al., 2008; Pollierer et al., 2009). Changes in d13C and d15N enrichment are considered to reflect changes in diet and are used to describe trophic status such as preferential feeding on fresh or humified materials. In this respect, the amount of C and d13C, and N and d15N, in casts, can be expected to reflect the earthworm’s ecological position or feeding strategy. In our study, d13C ratios of CAST aggregates had values in between those of roots and leaves, while CONTROL aggregates were more enriched in 13C and 15N. This confirms field observations and A. khami can be classified as anecic (sensu Bouché, 1977). Anecic earthworms mainly feed on litter and incorporate the undigested OM into its casts (Crow et al., 2009). The higher lignin content in the casts compared to the surrounding soil aggregates appears to support this observation. 4.3. Influence of earthworms on SOM dynamics Although earthworms initially speed up the breakdown of SOM (i.e., Coq et al., 2007), several studies suggested that they may also stabilize SOM in their casts in the long-term through the incorporation and protection of SOM into microaggregates within macroaggregates (Bossuyt et al., 2004; Pulleman et al., 2005; Fonte et al., 2007). Nevertheless opposite effects have also been observed in agroecosystems depending on the way they are managed (Pulleman et al., 2005; Fonte et al., 2007). These newly formed microaggregates protect SOM from decay, even after the breakdown of the macroaggregates in which they are formed. The influence of earthworms on SOM is usually appreciated by examining the evolution of C and N in soil aggregates, without seeking further information on the quality of the SOM. Measuring the total C and N content did not allow us to make conclusions about SOM speciation in soil aggregates of different sizes. This was achieved by analysis of lignin content and composition, which showed that relatively undecomposed lignin was present in the smaller size fractions. In particular, the significantly higher amounts of cinnamyl units in CAST microaggregates (50e250 mm) compared to bigger aggregates may be indicative of the preservation of relatively undecomposed lignin. Cinnamyl units are located at the periphery of the lignin molecule and most easily degraded by microbial attack (Bahri et al., 2006). Lignin is degraded rapidly in tropical environments and the lignin content of tropical soils is low, with the remaining lignin units showing a high degree of oxidation (Rumpel et al., 2008). Our results may therefore indicate that earthworm activity is essential to protect easily decomposable plant material through incorporation into small sized aggregates. However, it is worth noting that more mineral-bound C and N were lost for CONTROL samples than for CAST samples after HF treatment. This suggests a tighter association between OM and soil minerals in CONTROL aggregates and that A. khami activity did not lead to mineral bonding of a high proportion of easily soluble SOM compounds. Protection of SOM within casts of A. khami may therefore be explained more by their physical (e.g., higher bulk density and increased soil structural stability, Jouquet et al., 2008a), rather than chemical properties. We are aware of only one other article describing the evolution of the quality of lignin and carbohydrates in earthworm casts (Guggenberger et al., 1996). These authors analyzed the chemical composition of SOM in particle size fractions of earthworm casts and the surrounding soil and observed that slightly altered plant residues accumulate in earthworm casts produced by anecic earthworms. Although the way we produced soil aggregates (i.e., rainfall simulation followed by wet sieving) differed substantially from theirs (particle size separation by ultrasound), our results confirm the accumulation of slightly altered plant residues in earthworm casts produced by anecic earthworms. In addition,
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our results showed that plant material may be protected within the smallest water-stable aggregates composing the original cast. Analytical pyrolysis also allowed us to demonstrate that aggregate size influences SOM biogeochemical composition, for both CAST and CONTROL soil aggregates. Consistent with our previous interpretation of soil organization from C and N measurements, analytical pyrolysis showed that CAST and CONTROL samples <2000 mm could be clearly distinguished according to the molecular properties of their SOM. The distinction between smaller aggregates within CAST or CONTROL, however, was more difficult. These results confirm that the distinction between micro(<250 mm) and macroaggregates (>250 mm) is not relevant for an understanding of SOM organization in soil aggregates at our study site. Interestingly, we observed a high variation in the biogeochemical signature of CAST and CONTROL >2000 mm, which could indicate that they consist of numerous smaller aggregate size fractions plus other organic molecules that probably act as binding agents. 5. Conclusion This study provides evidence that the stoichiometric approach to quantify the magnitude and extent of earthworm activity on SOM chemistry has limitations. Molecular analyses clearly showed that (i) the biochemical composition of all CAST and CONTROL aggregate size classes <2000 mm is different and the (ii) protection of OM is most enhanced in the smallest aggregate size fraction of CAST. Our data further indicate that OM in all size fractions of CAST is relatively undecomposed material. Therefore, occlusion of OM into soil aggregates seems to be enhanced by earthworm activity rather than decomposition and subsequent protection of OM by soil minerals. Acknowledgments We would like to thank Pascal Podwojewski and Jerome Mathieu for constructive discussions. This project was supported financially by CNRS/INSU (VERAGREGAT project under the framework of the EC2CO program), IRD (unit research UMR-211-BIOEMCO) and CNRS (unit research UMR-7618-BIOEMCO) French institutes and the Management of Soil Erosion Consortium (MSEC) from the International Water Management Institute (IWMI). References Bahri, H., Dignac, M.-F., Rumpel, C., Rasse, D.P., Chenu, C., Mariotti, A., 2006. Lignin turnover kinetics in agricultural soils is monomer specific. Soil Biology and Biochemistry 38, 1977e1988. Baldock, J.A., Skjemstad, J.O., 2000. Role of the soil matrix and minerals in protecting natural organic materials against biological attack. Organic Geochemistry 31, 697e710. Barois, I., Villemin, G., Lavelle, P., Toutain, F., 1993. Transformation of the soil structure through Pontoscolex corethrus (Oligochaeta) intestinal tract. Geoderma 56, 57e66. Blanchart, E., Bruand, A., Lavelle, P., 1993. The physical structure of casts of Millsonia anomala (Oligochaeta: Megascolecidae) in shrub savanna soils (Côte d’Ivoire). Geoderma 56, 119e132. Blanchart, E., Albrecht, A., Alegre, J., Duboisset, A., Gilot, C., Pashanasi, B., Lavelle, P., Brussaard, L., 1999. Effects of earthworms on soil structure and physical properties. In: Lavelle, P., Brussaard, L., Hendrix, P. (Eds.), Earthworm Management in Tropical Agroecosystems. CAB International, pp. 149e171. Bossuyt, H., Six, J., Hendrix, P.F., 2004. Rapid incorporation of carbon from fresh residues into newly formed stable microaggregates within earthworm casts. European Journal of Soil Science 55, 393e399. Bossuyt, H., Six, J., Hendrix, P.F., 2005. Protection of soil carbon by microaggregates within earthworm casts. Soil Biology and Biochemistry 37, 251e258. Bouché, M.B., 1977. Stratégies lombriciennes. Ecological Bulletin 25, 122e132. Bracewell, J.M., Pacey, N., Robertson, G.W., 1987. Organic matter in onshore cretaceous chalks and its variations, investigated by pyrolysis-mass spectrometry. Journal of Analytical and Applied Pyrolysis 10, 199e213.
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impact of earthworms on soil and carbon erosion in steep slope ecosystem. A study case in Northern Vietnam. Catena 81, 113e116. Kögel, I., Bochter, R., 1985. Characterization of lignin in forest humus layers by highperformance liquid chromatography of cupric oxide oxidation products. Soil Biology and Biochemistry 17, 637e640. Lal, R., 2004. Soil carbon sequestration to mitigate climate change. Geoderma 123, 1e22. Lavelle, P., Spain, A.V., 2001. Soil Ecology. Kluwer Academic Publishers, Dordrecht, Netherlands, 654 pp. Neilson, R., Boag, B., Smith, M., 2000. Earthworm d13C and d15N analyses suggest that putative functional classifications of earthworms are site-specific and may also indicate habitat diversity. Soil Biology and Biochemistry 32, 1053e1061. Nierop, K.G.J., Pulleman, M.M., Marinissen, J.C.Y., 2001. Management induced organic matter differentiation in grassland and arable soil: a study using pyrolysis techniques. Soil Biology and Biochemistry 33, 755e764. Podwojewski, P., Orange, D., Jouquet, P., Valentin, C., Nguyen Van, T., Janeau, J.L., Duc Toan, T., 2008. Land-use impacts on surface runoff and soil detachment within agricultural sloping lands in Northern Vietnam. Catena 74, 109e118. Pollierer, M.M., Langel, R., Scheu, S., Maraun, M., 2009. Compartmentalization of the soil animal food web as indicated by dual analysis of stable isotope ratios (15N/14N and 13C/12C). Soil Biology and Biochemistry 41, 1221e1226. Pulleman, M.M., Six, J., Uyl, A., Marinissen, J.C.Y., Jongmans, A.G., 2005. Earthworms and management affect organic matter incorporation and microaggregate formation in agricultural soils. Applied Soil Ecology 29, 1e5. R Development Core Team, 2008. R: a Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, ISBN 3900051-07-0. Accessed at www.R-project.org. Rumpel, C., Rabia, N., Derenne, S., Quenea, K., Eusterhues, K., Kögel-Knabner, I., Mariotti, A., 2006. Alterations of soil organic matter following treatment with hydrofluoric acid (HF). Organic Geochemistry 37, 1437e1451. Rumpel, C., Chaplot, V., Chabbi, A., Largeau, C., Valentin, C., 2008. Stabilisation of HF soluble and HCl resistant organic matter in tropical sloping soils under slash and burn agriculture. Geoderma 145, 347e354. Saiz-Jimenez, C., De Leeuw, J.W., 1984. Pyrolysis-gas chromatography-mass spectrometry of isolated, synthetic and degraded lignins. Organic Geochemistry 10, 869e876. Scheu, S., Schlitt, N., Tiunov, A.V., Newington, J.E., Jones, T.H., 2002. Effects of the presence and community composition of earthworms on microbial community functioning. Oecologia 133, 254e260. Shipitalo, M.J., Protz, R., 1989. Chemistry and micromorphology of aggregation in earthworm casts. Geoderma 45, 357e374. Six, J., Elliott, E.T., Paustian, K., 2000. Soil macroaggregate turnover and microaggregate formation: a mechanism for C sequestration under no-tillage agriculture. Soil Biology and Biochemistry 32, 2099e2103. Six, J., Conant, R.T., Paul, E.A., Paustian, K., 2002. Stabilization mechanisms of soil organic matter: implications for C-saturation of soils. Plant and Soil 241, 155e176. Six, J., Bossuyt, H., Degryze, S., Denef, K., 2004. A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics. Soil and Tillage Research 79, 7e31. Tisdall, J.M., Oades, J.M., 1982. Organic matter and water-stable aggregates in soils. Journal of Soil Science 33, 141e163. Valentin, C., Agus, F., Alamban, R., Boosaner, A., Bricquet, J.P., Chaplot, V., de Guzman, T., de Rouw, A., Janeau, J.L., Orange, D., Phachomphonh, K., Do Duy, P., Podwojewski, P., Ribolzi, O., Silvera, N., Subagyono, K., Thiébaux, J.P., Tran Duc, T., Vadari, T., 2008. Runoff and sediment losses from 27 upland catchments in Southeast Asia: impact of rapid land use changes and conservation practices. Agriculture Ecosystems and Environment 128, 225e238. Weil, R.R., Magdoff, F., 2004. Significance of soil organic matter to soil quality and health. In: Magdoff, F., Weil, R.R. (Eds.), Soil Organic Matter in Sustainable Agriculture. CRC Press, pp. 1e44. WRB: World Reference Base for soil resources, 2006. World Soil Resources Reports, N 103. FAO, Rome, 145 pp.
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How do earthworms influence organic matter quantity and quality in tropical soils? Hanh Nguyen Hong a, b, Cornelia Rumpel c, Thierry Henry des Tureaux a, b, Gérard Bardoux c, Daniel Billou c, Toan Tran Duc a, Pascal Jouquet a, b, * a b c
IRD e IWMI e SFRI, Dong Ngac, Tu Liem, Hanoï, Viet Nam IRD, UMR 211 BIOEMCO, Centre IRD Ile de France, Bondy, France CNRS, UMR 7618 BIOEMCO, Campus Paris AgroTech, Thiverval-Grignon, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 June 2010 Received in revised form 22 September 2010 Accepted 24 September 2010 Available online 9 November 2010
Earthworms are important regulators of soil structure and soil organic matter (SOM) dynamics; however, quantifying their influence on SOM cycling in tropical ecosystems remains little studied. Simulated rainfall was used to disrupt casts produced by Amynthas khami and their surrounding soil (control) into a range of small sized aggregates (50e250, 250e500, 500e2000 and 2000e5000 mm). To gain insight into how earthworms influence SOM biogeochemical composition in the aggregates, we carried out elemental and stable isotope analysis, and analytical pyrolysis (Py GC/MS). We also characterized their lignin component after oxidation with cupric oxide (CuO). The C content of smaller size fractions (<500 mm) in the control soil was higher than in the larger fractions. Our study therefore suggests that the aggregate hierarchy concept, which is used to understand soil aggregates and SOM dynamics in temperate soils, may not be applicable to the tropical Acrisol studied here. Earthworms modified SOM organization in soil aggregates. Although the isotope analyses were useful for highlighting SOM enrichment in the earthworm casts, aggregate fractions could not be classified according to particle size. Molecular analyses were necessary to indicate that SOM in all size fractions of casts consisted of relatively undecomposed material. Protection of the most labile SOM structures occurred in the smallest aggregate size fraction (50e250 mm). Py GC/MS showed that earthworm casts and control aggregates <2000 mm could be clearly distinguished according to the molecular properties of their SOM. Aggregates larger than 2000 mm, however, were most probably composed of all fractions and were not different. As a consequence, our results indicate that studies to determine the impact of earthworms on SOM turnover in soil are spatially dependant on the scale of observation. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Earthworms SOM Soil aggregate organization Pyrolysis GC/MS Lignin
1. Introduction Soil organic matter (SOM) is an important active carbon reservoir at the global scale and plays a pervasive role in promoting soil ecological functions. By contributing greatly to a number of soil properties including soil structure, porosity, water retention, cationic exchange and pH buffering capacities, SOM is a fundamental component, ensuring soil fertility (Lal, 2004; Weil and Magdoff, 2004). Understanding the dynamics of SOM is therefore of primary importance and requires knowledge of the factors affecting its stability. Soil aggregates have been proposed as
* Corresponding author. IRD, UMR 211 BIOEMCO, Centre IRD Ile de France, Bondy, France. E-mail address: [email protected] (P. Jouquet). 0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.09.033
structural units within the soil that control the dynamics of SOM and nutrient cycling (e.g., Tisdall and Oades, 1982; Chevallier et al., 2004). They are usually considered as organized units in the form of micro- (50e250 mm) and macroaggregates (>250 mm) occluding particulate organic matter (POM) (see Six et al., 2004 for a review). Several studies focused on the mechanisms by which earthworms influence SOM sequestration within soil aggregates (See a review in Lavelle and Spain, 2001). Soil aggregates are completely reorganized after their passage through earthworm guts (Shipitalo and Protz, 1989; Barois et al., 1993). SOM is partially digested by earthworms and one proportion becomes physically protected in microaggregates (i.e., Bossuyt et al., 2004, 2005; Fonte et al., 2007). This physical protection is explained by the higher water stability of casts compared to the surrounding soil, because: (i) microbial polysaccharides and other organic products strengthen bonds between organic and mineral components (Shipitalo and Protz,
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1989); (ii) casts have higher bulk density and/or the presence of a cortex on their periphery which renders them impermeable to water (Blanchart et al., 1993, 1999; Jouquet et al., 2008a). Most of the studies dealing with the influence of earthworms on soil properties have focused on the total C and N content, and the subsequent C:N ratio of SOM as an indicator of soil quality (i.e., Desjardins et al., 2003; Coq et al., 2007; Eisenhauer et al., 2007; Fonte et al., 2007). More recently, stable isotopes were also used to estimate the trophic position of earthworms or the origin of SOM in their casts (i.e., Bossuyt et al., 2004, 2005; Fonte et al., 2007; Don et al., 2008; Hyodo et al., 2008; Pollierer et al., 2009). Although measuring total C and N content might be interesting for estimating the impact of earthworms on the C budget at the ecosystem scale, it can also been considered as non-exhaustive and does not contribute to a clear understanding of how earthworms influence SOM mineralization and sequestration. Molecular based information on OM chemistry may provide additional information on this subject (Guggenberger et al., 1996). In this study we focused on “giant” earthworm casts that are very commonly produced in the tropics (Fig. 1). Our objectives were (i) to assess the relevance of the stoichiometric approach to describe SOM quality through its C and N content, and (ii) to evaluate what information can be gained from a biogeochemical analysis of aggregate size fractions concerning the effect of earthworm activity on SOM composition and its protection.
2. Material and methods 2.1. Study site and study model Soil and cast aggregates were sampled in the experimental catchment (46 ha) of the MSEC (Management Soil Erosion Consortium of the International Water Management Institute, IWMI) project (Valentin et al., 2008). This study site is located in Dong Cao village, in north-eastern Vietnam, approximately 50 km south-west of Hanoi (20 570 N, 105 290 E). The annual rainfall ranges from 1500 to 1800 mm, of which 80e85% occurs from April to October. The air humidity is always high, between 75 and 100%. The mean daily temperature varies from 15 C to 25 C. The soil is an Acrisol (WRB, 2006) with more than 50% clay, mainly kaolinite, with a low pH of around 5, and a low CEC (<10 cmol kg soil1) (Jouquet et al., 2008a,b; Podwojewski et al., 2008). Earthworm casts were produced by Amynthas khami. A. khami varies in size and adults can reach up to more than 50 cm in length. It builds casts that are partly deposited on the soil surface (Fig. 1) but does not influence soil particle size (around 66% clay, 27.5% silt and 6.4% sand, Jouquet et al., 2007). Casts are globular and characterized by a very high soil structural stability (Jouquet et al., 2008a). The abundance of A. khami in the experimental watershed was previously reported by Jouquet et al. (2008b) and reaches 1.11 (0.59) ind m2. 2.2. Soil sampling
Fig. 1. Casts produced by Amynthas khami. Fecal aggregates are deposited one on top of the other [photo, P. Jouquet, 2009].
Soil and cast aggregates were collected in a fallow field following a Eucalyptus plantation which was cut down in 2003. In this area, there was no soil preparation and no herbicides were applied but the vegetation cover was maintained at a low level (<50 cm) by livestock grazing. Casts (CAST) were collected on the soil surface and control soil aggregates (CONTROL) were sampled in three different randomly chosen areas in the fallow. The number of repetitions was therefore n ¼ 3. CAST and CONTROL constitute the two dominant free soil surface aggregates which are fragmented by raindrop impacts at the beginning of the rainy season (Jouquet et al., 2010). For that, samples were taken few days before the rainy season, in March 2008. CAST were collected on the soil surface. They were carefully selected according to their aspect and only rounded shape aggregates without trace of alteration by the rain were sampled. A previous study showed that CAST lose their rounded shape aspect very rapidly and get an angular aspect when affected by the rain (Jouquet et al., 2009). We assumed that this sampling design allowed us to sample dry CAST which had approximately the same history (i.e., they had been produced several months before, probably at the end of the previous rainy season in October 2007). Microbial activity is higher in freshly emitted earthworm casts and then decreases with time (Lavelle and Spain, 2001). Sampling soil aggregates at the end of the dry season after several months of incubation therefore also allowed us to reduce the variability which is inherent at any soil sampling. In March 2008, tree seedlings were planted in the fallow and many dry soil clods (>5000 mm) formed during soil preparation (no tillage but shallow holes with a spade) were deposited on the soil surface. These soil aggregates were carefully sampled and inspected to be sure that there was no trace of earthworm activity (casts or galleries) and were considered as CONTROL. Soil samples were air-dried for three days. In addition, a large amount fresh leaves were randomly sampled from different non-identified plant species in each plot within the studied area. The samples were airdried for three days and thoroughly mixed to produce three composite samples (one per plot). A similar procedure was applied for root sampling.
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2.3. Rainfall simulation Simulated rainfall was used to fragment soil aggregates. The rainfall intensity was 44 mm h1 (i.e., kinetic energy w268 J m2), which was estimated to be equivalent to a medium intensity rainfall event. About 80 g of CAST aggregates, corresponding to approximately one individual earthworm cast, or 80 g of CONTROL aggregates were put on a 500 mm sized mesh grid (15 15 cm) which covered a 10 L bucket in order to recover water and aggregates that passed through the grid. Because CAST are more stable than CONTROL aggregates (Jouquet et al., 2008a), aggregates which remained on the grid were collected after 1 and 2 h of rainfall simulation, respectively for CONTROL and CAST aggregates. Soil aggregates remaining on the grid were gently sieved in distilled water and carefully separated into 4 size classes: 50e250, 250e500, 500e2000 and 2000e5000 mm. These different fractions were air-dried for 2 days. All the procedures were repeated for each sampled area (n ¼ 3). 2.4. Elemental and stable isotope analyses The organic carbon (C) and nitrogen (N) content of each aggregate size class were determined by the dry combustion method using a CHN elemental analyzer (CHN NA 1500, Carlo Erba). Analytical precision was 0.1 mg g1 for C and 0.05 mg g1 for N content. The C and N isotope ratios of aggregate size classes of CAST and CONTROL aggregates were compared to those of roots and leaves. Stable isotope ratios were determined with an elemental analyzer (EA, Eurovector) coupled to an isotope ratio mass spectrometer in continuous flow mode (Isoprime GV instruments). C and N isotope ratios were defined using the delta notation:
dsample (&) ¼ [(Rsample Rstandard)/Rstandard] 1000 where R is the isotopic ratio 13C/12C and 15N/14N for the sample and reference gas, Rsample and Rstandard, respectively. The stable isotope ratios were calculated relative to the Pee Dee Belemnite standard (PDB) for C and relative to atmospheric N2 for N. Analytical precision for isotope measurements was 0.1& for C and 0.2& for N isotopes. 2.5. Mineral-bound OM We assessed the amount of mineral-bound OM, which is usually protected against microbial attack (Baldock and Skjemstad, 2000) after mineralization of the sample using hydrofluoric acid (HF). This treatment was found to isolate mineral-bound carbon without affecting the OM composition of the remaining material (Rumpel et al., 2006). The HF treatment was performed on aggregate size fractions by adding 10 ml of 10% HF to 2 g ground mineral soil material (Eusterhues et al., 2007). The suspensions were shaken for 2 h at room temperature, then centrifuged and the supernatant removed. This procedure was repeated five times. Then the residues were washed five times with deionised H2O to remove salts and residual HF. Afterwards the samples were freeze-dried and weighed. The concentration of organic carbon (OC) was determined and the amount of OC lost upon this treatment was calculated by mass balance calculation. 2.6. Lignin content and composition The lignin content and composition of the samples were determined through release of the phenol monomers by alkaline CuO oxidation (Hedges and Ertel, 1982). This method allows both
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parameters to be assessed quantitatively, which gives an indication of (i) the presence of intact plant material and (ii) the state of OM decomposition. Briefly, 50 mg of pure organic matter and 500 mg of the mineral A horizon were oxidized with 250 mg CuO and 2 M NaOH at 172 C under N2 for 2 h. After purification using solidphase extraction with a C18 column (International Sorbent Technology) as suggested by Kögel and Bochter (1985), lignin phenols were derivatised and quantified as trimethylsilyl derivatives by gas chromatography. We used a HP GC 6890 gas chromatograph equipped with a flame ionisation detector. Separation of the individual monomers was achieved with a SGE BPX-5 column (50 m length, 0.25 mm inner diameter, 0.32 mm coating). The temperature was programmed from 100 C (1 min isotherm) to 150 C at 3 C min1 and from 150 to 300 C at 1 C min1. The detector temperature was kept at 350 C. Helium was used as carrier gas (1 ml min1). This method yields a suite of phenolic CuO oxidation products, such as vanillyl (V), syringyl (S), and cinnamyl (C) compounds with aldehydic, ketonic, and acidic side chains. The sum of CuO oxidation products (VSC) is usually taken as an indicator of the total amount of lignin and ratios between the acidic and the aldehydic forms of the vanillyl and syringyl compounds (Ac/ Al)V,S indicate their degree of decomposition (Hedges et al., 1988). VSC is decreasing and the (Ac/Al)V,S are usually increasing with increasing OM degradation. C- and S-units are less stable than V units and therefore decreasing C/V as well as S/V ratios indicate more advanced stages of OM decay (Bahri et al., 2006). Analytical precision was 10% for VSC and 5% for ratios between phenolic products. 2.7. Analytical pyrolysis Analytical pyrolysis consists of curie-point pyrolysis of OM and identification of pyrolysis products using gas chromatography coupled to mass spectrometry (Py GC/MS). It gives a good overview of the molecular composition of the bulk material in terms of the contribution of polysaccharides, N-containing, lignin-derived and aliphatic compounds. Curie-point pyrolysis was carried out with a pyrolysis unit (GSG Curie-point Pyrolyser 1040 PSC) coupled to a gas chromatograph (Hewlett Packard HP 5890) and a mass spectrometer (Hewlett Packard HP 5889; electron energy 70 eV). Approximately 0.5e1 mg of HF-treated sample were loaded in tubular ferromagnetic wires and inductively heated to their Curie temperature at 650 C during 0.15 s in the pyrolysis unit. The pyrolysis products were transferred to the GC system using the splitless mode with He as the carrier gas. The pyrolysis products were separated on a 60 m fused silica capillary column with a wax stationary phase (SolGelWax, SGE, 0.32 mm i.d., film thickness 0.5 mm). The temperature of the GC oven was programmed from 30 C to 280 C at 2 C min1. The final temperature was held for 15 min. We chose a polar column, because we were interested in the relative quantification of polysaccharide-derived and ligninderived compounds as well as N-containing compounds from the GC traces. These compounds are most likely constituting the bulk of SOM in tropical clayey soils (Rumpel et al., 2006). Such compounds were found to yield broad peaks when separated on a non-polar column (Dignac et al., 2005). We identified five types of pyrolysis products according to their origin: (1) polysaccharide-derived compounds; (2) lignin-derived compounds; (3) aliphatic compounds derived from lipids or other aliphatic biomacromolecules; (4) N-containing compounds most likely derived from proteins or other N-containing molecules and (5) non-specific compounds derived from more than one of the other substance classes. The peak areas of the Total Ion Current (TIC) trace were integrated using the GC ChemStation program
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(Agilent Technologies). The total area of the five types of pyrolysis products was calculated as the sum of the identified peaks and the relative contribution of each compound class to this sum was reported.
Table 1 Repeated measures ANOVA table for the effect of soil aggregate origin (CAST vs. CONTROL) and size (50e250, 250e500, 500e2000 and 2000e5000 mm) on the C and N content and C and N isotope ratios. F-values are displayed. ***p < 0.001, **p < 0.01, *p < 0.05, ns p > 0.05. C
2.8. Statistical analyses Before analyses, data were tested for homogeneity of variance using the Levene’s test. Data were analyzed by repeated measures analysis of variance (ANOVA) with the origin of the soil aggregates (CAST vs. CONTROL) and the soil aggregate sizes (50e250, 250e500, 500e2000 and 2000e5000 mm) as the independent variables and the aggregate origin as within-subject factor. Comparisons between means were tested with LSD test. A Principal Component Analysis (PCA) was carried out to differentiate the samples based on their pyrolysis signature. Briefly, we used a matrix of 24 samples and the relative contribution of the five types of pyrolysis products previously described. All statistical calculations were carried out using R (R Development Core Team, 2008). Differences among treatments were declared at the p < 0.05 probability level of significance. 3. Results 3.1. SOM content and stable isotope composition The organic C and N content in soil aggregates are shown in Fig. 2 and results of the statistical analyzes are shown in Table 1. CAST aggregates were characterized by a higher C and N content than CONTROL aggregates for all the aggregate size classes. C and N contents were not influenced by aggregate size for CAST.
Origin (1) Aggregate size (2) (1) (2)
N
d13C
d15N
df
F
F
F
F
1 3 3
123.8*** 5.3* 2.7 ns
109.5*** 3.6* 2.9 ns
95.0*** 17.1*** 15.1***
55.7*** 1.8 ns 7.2**
Conversely, CONTROL soil aggregates smaller than 500 mm had significantly more C and N than those bigger than 500 mm. Fig. 3 shows the d13C and d15N in CAST, CONTROL and in the composite samples of roots and leaves. Stable isotope ratios were dependent on soil origin and aggregate sizes (Table 1). Interestingly, the smaller size fractions were depleted in 13C and 15N compared to the bigger size fractions. CAST samples were characterized by a signature closer to that of leaves and roots compared to CONTROL samples, which generally showed higher 13C and 15N enrichment. d13C in CASTaggregates showed increased 13C enrichment compared to leaves and 13C depletion compared to roots and CONTROL aggregates. The d15N signatures of both types of aggregates were enriched in 15N with regards to the plant material. CAST aggregates smaller than 250 mm had a significantly different d13C and d15N signatures compared to those larger than 250 mm. For CONTROL samples, although no significant differences were observed for aggregates between 50e250 mm and 250e500 mm, aggregates between 500e2000 mm and >2000 mm had specific d13C and d15N signatures. 3.2. Mineral-bound OM Demineralization of soil samples by treatment with hydrofluoric acid led to a significantly higher reduction in soil weight for CONTROL compared with CAST aggregates (93.5 vs. 90.6%, SE: 0.18 and 0.15, for CONTROL and CAST aggregates, respectively; Table 2), without a significant effect of aggregate size. As a consequence, more mineralbound C was lost for CONTROL samples than for CAST samples, whatever the aggregate sizes (Fig. 4). For both CAST and CONTROL, more important C losses were observed for aggregates larger than 2000 mm, and no difference were found between the other aggregate size classes. The same trend was observed with N loss.
Fig. 2. Total C and N content (%) in cast (CAST, in black) and control (CONTROL, in white) soil aggregates according to their sizes (>2000, 2000e500, 500e250, 250e50 mm). Histograms with the same letters are not significantly different at p ¼ 0.05. Bars represent the standard errors.
Fig. 3. d13C and d13N values for leaves and roots (triangle), casts (sphere) and control (square) soil aggregates of different size classes (>2000, 2000e500, 500e250, 250e50 mm). Data within the same ellipse are not significantly different at p ¼ 0.05. Bars represent the standard errors.
H.N. Hong et al. / Soil Biology & Biochemistry 43 (2011) 223e230 Table 2 Repeated measures ANOVA table for the effect of soil aggregate origin (CAST vs. CONTROL) and size (50e250, 250e500, 500e2000 and 2000e5000 mm) on the amount of soil weight and C and N loss after hydrofluoric acid treatment. F-values are displayed. ***p < 0.001, *p < 0.05, ns p > 0.05. Soil loss
Origin (1) Aggregate size (2) (1) (2)
C loss
N loss
df
F
F
F
1 3 3
35.3 *** 0.2 ns 1.4 ns
24.5*** 28.3*** 3.6*
7.4* 21.3*** 17.5***
3.3. Lignin analyses The total lignin contribution to SOM content, estimated by the sum of vanillyl (V), syringyl (S) and cinnamyl (C) units (VSC), was significantly higher in CAST than in CONTROL samples <2000 mm without a significant influence of aggregate size (Table 3). Conversely, CAST >2000 mm had similar VSC content than all the CONTROL aggregate size classes. CAST aggregates were characterized by a higher amount of C units compared to the CONTROL samples, whereas no difference was observed in the amount of V and S units. C units in CAST had significantly higher concentrations in microaggregates (50e250 mm) compared to bigger aggregates and their CONTROL counterparts. The acid to aldehyde ratio of vanillyl (Ac/Al)v was higher in CONTROL samples >2000 mm than in CAST of the same size, without any significant difference for the other aggregate size fractions. This is in line with the elevated C/V and S/V ratios in CAST >2000 mm compared to CONTROL >2000 mm and could indicate that lignin in the CONTROL samples was more degraded than in CAST samples. No significant differences were observed for (Ac/Al)s between CAST and CONTROL aggregates of the same size.
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3.4. Assessment of SOM quality by analytical pyrolysis (Py GC/MS) From the total ion chromatograms, a number of pyrolysis products were identified, which were derived from five different biochemical families. All pyrograms showed peaks related to polysaccharide-derived, lignin-derived, N-containing, aliphatic and non-specific compounds (data not shown) (Fig. 5). Pyrolysis products with a furan, cyclopentene and cyclopropane structure were identified as the main polysaccharide-derived compounds. Ligninderived compounds were dominated by methoxy phenols, which indicate the contribution of guaiacyl lignins (Nierop et al., 2001). Consistent with the results obtained by CuO oxidation, the occurrence of pyrolysis products based on dimethoxy phenols indicates the presence of syringyl lignin. N-containing compounds include pyrrols and pyridines. Pyrrol and its derivatives are formed during the pyrolysis of proteins by cyclization (Chiavari and Galletti, 1992), but may also be pyrolysis products of chlorophyll (Bracewell et al., 1987). Additionally, many non-specific compounds were released during pyrolysis of the three soils. Acetic acid, phenol, methyl phenols, styrene and toluene are non-specific compounds, since they are derived from the pyrolysis of protein, polysaccharides, lignins peptides and tannins (Saiz-Jimenez and Leeuw, 1984). For CAST samples, a large number of lignin-derived pyrolysis products and fewer N-containing compounds contributed to the total pyrolysis products identified. Principal Component Analysis (PCA) of the relative contribution of the five types of pyrolysis products is shown in Fig. 6. Axis 1 and 2, respectively, explained 44.4 and 25.2% of the total data variance. Axis 1 is mostly determined by the contribution of N-containing and polysaccharide-derived compounds. The PCA plot of the samples in the plane shows that CAST and CONTROL aggregates <2000 mm from the same size class were clearly separated on the first and second axis according to their biogeochemical composition as seen by Py GC/MS. The molecular composition of CAST aggregates did not allow us to clearly distinguish between aggregates <2000 mm within the CAST and CONTROL samples. In addition, aggregates >2000 mm were characterized by a high variance and overlapped all other size fractions. 4. Discussion 4.1. Relevance of the soil aggregate hierarchy in Acrisols
Fig. 4. Total amount of C and N lost (%) after treatment of the samples (earthworm casts in black; control soil in white) with hydrofluoric acid. Histograms with the same letters are not significantly different at p ¼ 0.05. Bars represent the standard errors.
Soil aggregate organization is usually described by comparing micro- and macroaggregrate soil properties (see Six et al., 2004 for a review). Microaggregates (50e250 mm) are especially important in regulating SOM dynamics. They are characterized by a high structural stability, which results in a low rate of SOM turnover. Conversely, macroaggregates (250e2000 mm) are less stable and the SOM turnover rate increases (Six et al., 2000, 2002). However, aggregate hierarchy and the relevance of micro- and macroaggregate classification for understanding SOM dynamics have limitations in highly weathered tropical soils, such as Oxisols (Zoratelli et al., 2005, 2007). We are not aware of any studies that describe C and N pools in micro- and macroaggregates in Acrisols. In our study, the highest SOM content in small size aggregates (<500 mm) contradicts the concept of aggregate hierarchy presented by Tisdall and Oades (1982), who suggested that larger aggregates are more enriched in SOM than smaller aggregates. However, we did confirm that a distinction exists between small and larger size aggregates due to their SOM content, with an alternative functional limit observed at 500 mm and not 250 mm, as usually observed in temperate soils (Six et al., 2004). We assume that this might be explained by the ability of Acrisols to produce water-stable pseudosilts and pseudo-sand aggregates due to the high reactivity of
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Table 3 Lignin contribution to SOM content (mg gC1), estimated by the sum of vanillyl (V), syringyl (S) and cinnamyl (C) units (VSC) and ratios between the acidic and the aldehydic forms of the vanillyl and syringyl compounds (Ac/Al)V,S in CAST and CONTROL samples according to their sizes (>2000, 2000e500, 500e250, 250e50 mm). Standard errors in parenthesis, n ¼ 3. F-values of repeated measures ANOVA on the effect of soil aggregate “origin” (CAST or CONTROL) and “size” (50e250, 250e500, 500e2000 and >2000 mm) factors and their interactions. ***p < 0.001, **p < 0.01, *p < 0.05, ns p > 0.00.5. V
S
C
CAST >2000 mm 2000e500 mm 500e250 mm 250e50 mm
3.14 6.18 5.05 7.12
(0.37) (1.37) (1.25) (1.66)
2.78 4.59 3.35 4.57
(0.43) (0.93) (0.92) (1.05)
1.64 2.79 3.00 5.12
CONTROL >2000 mm 2000e500 mm 500e250 mm 250e50 mm
4.59 5.15 4.36 2.68
(1.70) (1.42) (0.52) (0.08)
1.89 3.82 2.23 1.74
(0.62) (1.32) (0.15) (0.01)
1.35 2.36 1.85 1.61
F-values Origin Size Origin*Size
0.8 ns 0.8 ns 2.1 ns
1.4 ns 2.0 ns 0.8 ns
VSC
S/V
(0.30) (0.41) (0.61) (1.04)
7.56 (0.25) 13.56 (2.53) 11.40 (2.78) 16.83 (3.70)
0.95 0.76 0.65 0.65
(0.27) (0.11) (0.04) (0.04)
0.52 0.47 0.61 0.73
(0.04) (0.04) (0.03) (0.04)
0.65 0.64 0.93 1.13
(0.48) (0.79) (0.09) (0.06)
7.83 (2.79) 11.33 (3.41) 8.44 (0.62) 6.03 (0.05)
0.42 0.76 0.52 0.65
(0.03) (0.13) (0.04) (0.02)
0.29 0.46 0.44 0.60
(0.01) (0.06) (0.06) (0.04)
0.71 0.62 0.84 0.92
1.6 ns 1.5 ns 2.4 ns
0.4 ns 0.6 ns 2.0 ns
2.8 ns 3.8* 3.6*
oxides, and/or the method we used to produce the aggregate size classes (rainfall simulation plus wet sieving in our study vs. wet sieving alone in the other studies). Acrisols are tropical and subtropical soils, which are less weathered than Oxisols (i.e., Ferralsols; WRB, 2006). Further studies are therefore needed to confirm our observation, i.e., the evolution of micro-aggregate sizes in Acrisols, and to determine the limitations and magnitude of the aggregate hierarchy hypothesis. Conversely in casts, the C and N content did not vary according to the aggregate sizes, suggesting that those made by earthworms (CAST) had a homogeneous organization. This finding is in agreement with the fact that casts produced by A. khami are described as an accumulation of small size aggregates <500 mm into bigger size aggregates (>2000 mm), while the control surrounding soil is characterized by a hierarchical organization of smaller aggregates (<500 mm) inside larger water-stable aggregates (>500 mm) (Jouquet et al., 2009).
C/V
C/S
1.4 ns 12.4*** 2.0 ns
(Ac/al)v
(Ac/al)s
(0.20) (0.09) (0.07) (0.11)
0.31 0.86 0.47 0.56
(0.08) (0.21) (0.07) (0.11)
0.83 0.60 0.45 0.45
(0.30) (0.16) (0.04) (0.04)
(0.03) (0.04) (0.05) (0.03)
1.00 0.75 0.79 0.58
(0.17) (0.09) (0.09) (0.06)
0.76 0.57 0.53 0.54
(0.28) (0.06) (0.02) (0.01)
0.3 ns 6.5 ** 0.6 ns
0.1 ns 1.6 ns 4.9*
0.4 ns 4.2* 0.3 ns
A. khami, which involves consuming fresh organic debris on and/or in soil. The use of stable isotope techniques is a useful complementary method for exploring the feeding ecology of earthworm field populations (Neilson et al., 2000; Scheu et al., 2002; Curry and
4.2. SOM content and quality as an indicator of earthworm ecological niche Classically, earthworm casts are said to be enriched in SOM compared to their surrounding soil environment (Lavelle and Spain, 2001). Our study confirms this general trend and C and N contents were significantly higher in earthworm casts than the surrounding soil. This is because of the feeding behavior of
Fig. 5. Proportion of pyrolysis products identified in CAST and CONTROL samples. POLYSAC: polysaccharide-derived compounds; LIG: lignin-derived compounds; ALIPHATIC: aliphatic compounds; N: N-containing compounds; NON-SPECIFIC: nonspecific compounds.
Fig. 6. Principal components analysis (PCA) carried out with the organic matter quality measured by Pyrolise in casts (CAST) and control (CONTROL) soil aggregates according to their sizes (>2000, 2000e500, 500e250, 250e50 mm). (a) Eigenvalue diagram. (b) Correlation circle on F1eF2 plane. Variables are polysaccharide-derived compounds (POLYSAC), lignin-derived compounds (LIG), aliphatic compounds derived from lipids or other aliphatic biomacromolecules (ALIPHATIC), N-containing compounds most likely derived from proteins or other N-containing molecules (N) and unspecific compounds derived from more than one of the other substance classes (NONSPECIFIC). (c) Soil structure ordered in the plane defined by factors 1 and 2. Ellipses represent 90% confidence limits.
H.N. Hong et al. / Soil Biology & Biochemistry 43 (2011) 223e230
Schmidt, 2007; Hyodo et al., 2008; Pollierer et al., 2009). Changes in d13C and d15N enrichment are considered to reflect changes in diet and are used to describe trophic status such as preferential feeding on fresh or humified materials. In this respect, the amount of C and d13C, and N and d15N, in casts, can be expected to reflect the earthworm’s ecological position or feeding strategy. In our study, d13C ratios of CAST aggregates had values in between those of roots and leaves, while CONTROL aggregates were more enriched in 13C and 15N. This confirms field observations and A. khami can be classified as anecic (sensu Bouché, 1977). Anecic earthworms mainly feed on litter and incorporate the undigested OM into its casts (Crow et al., 2009). The higher lignin content in the casts compared to the surrounding soil aggregates appears to support this observation. 4.3. Influence of earthworms on SOM dynamics Although earthworms initially speed up the breakdown of SOM (i.e., Coq et al., 2007), several studies suggested that they may also stabilize SOM in their casts in the long-term through the incorporation and protection of SOM into microaggregates within macroaggregates (Bossuyt et al., 2004; Pulleman et al., 2005; Fonte et al., 2007). Nevertheless opposite effects have also been observed in agroecosystems depending on the way they are managed (Pulleman et al., 2005; Fonte et al., 2007). These newly formed microaggregates protect SOM from decay, even after the breakdown of the macroaggregates in which they are formed. The influence of earthworms on SOM is usually appreciated by examining the evolution of C and N in soil aggregates, without seeking further information on the quality of the SOM. Measuring the total C and N content did not allow us to make conclusions about SOM speciation in soil aggregates of different sizes. This was achieved by analysis of lignin content and composition, which showed that relatively undecomposed lignin was present in the smaller size fractions. In particular, the significantly higher amounts of cinnamyl units in CAST microaggregates (50e250 mm) compared to bigger aggregates may be indicative of the preservation of relatively undecomposed lignin. Cinnamyl units are located at the periphery of the lignin molecule and most easily degraded by microbial attack (Bahri et al., 2006). Lignin is degraded rapidly in tropical environments and the lignin content of tropical soils is low, with the remaining lignin units showing a high degree of oxidation (Rumpel et al., 2008). Our results may therefore indicate that earthworm activity is essential to protect easily decomposable plant material through incorporation into small sized aggregates. However, it is worth noting that more mineral-bound C and N were lost for CONTROL samples than for CAST samples after HF treatment. This suggests a tighter association between OM and soil minerals in CONTROL aggregates and that A. khami activity did not lead to mineral bonding of a high proportion of easily soluble SOM compounds. Protection of SOM within casts of A. khami may therefore be explained more by their physical (e.g., higher bulk density and increased soil structural stability, Jouquet et al., 2008a), rather than chemical properties. We are aware of only one other article describing the evolution of the quality of lignin and carbohydrates in earthworm casts (Guggenberger et al., 1996). These authors analyzed the chemical composition of SOM in particle size fractions of earthworm casts and the surrounding soil and observed that slightly altered plant residues accumulate in earthworm casts produced by anecic earthworms. Although the way we produced soil aggregates (i.e., rainfall simulation followed by wet sieving) differed substantially from theirs (particle size separation by ultrasound), our results confirm the accumulation of slightly altered plant residues in earthworm casts produced by anecic earthworms. In addition,
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our results showed that plant material may be protected within the smallest water-stable aggregates composing the original cast. Analytical pyrolysis also allowed us to demonstrate that aggregate size influences SOM biogeochemical composition, for both CAST and CONTROL soil aggregates. Consistent with our previous interpretation of soil organization from C and N measurements, analytical pyrolysis showed that CAST and CONTROL samples <2000 mm could be clearly distinguished according to the molecular properties of their SOM. The distinction between smaller aggregates within CAST or CONTROL, however, was more difficult. These results confirm that the distinction between micro(<250 mm) and macroaggregates (>250 mm) is not relevant for an understanding of SOM organization in soil aggregates at our study site. Interestingly, we observed a high variation in the biogeochemical signature of CAST and CONTROL >2000 mm, which could indicate that they consist of numerous smaller aggregate size fractions plus other organic molecules that probably act as binding agents. 5. Conclusion This study provides evidence that the stoichiometric approach to quantify the magnitude and extent of earthworm activity on SOM chemistry has limitations. Molecular analyses clearly showed that (i) the biochemical composition of all CAST and CONTROL aggregate size classes <2000 mm is different and the (ii) protection of OM is most enhanced in the smallest aggregate size fraction of CAST. Our data further indicate that OM in all size fractions of CAST is relatively undecomposed material. Therefore, occlusion of OM into soil aggregates seems to be enhanced by earthworm activity rather than decomposition and subsequent protection of OM by soil minerals. Acknowledgments We would like to thank Pascal Podwojewski and Jerome Mathieu for constructive discussions. This project was supported financially by CNRS/INSU (VERAGREGAT project under the framework of the EC2CO program), IRD (unit research UMR-211-BIOEMCO) and CNRS (unit research UMR-7618-BIOEMCO) French institutes and the Management of Soil Erosion Consortium (MSEC) from the International Water Management Institute (IWMI). References Bahri, H., Dignac, M.-F., Rumpel, C., Rasse, D.P., Chenu, C., Mariotti, A., 2006. Lignin turnover kinetics in agricultural soils is monomer specific. Soil Biology and Biochemistry 38, 1977e1988. Baldock, J.A., Skjemstad, J.O., 2000. Role of the soil matrix and minerals in protecting natural organic materials against biological attack. Organic Geochemistry 31, 697e710. Barois, I., Villemin, G., Lavelle, P., Toutain, F., 1993. Transformation of the soil structure through Pontoscolex corethrus (Oligochaeta) intestinal tract. Geoderma 56, 57e66. Blanchart, E., Bruand, A., Lavelle, P., 1993. The physical structure of casts of Millsonia anomala (Oligochaeta: Megascolecidae) in shrub savanna soils (Côte d’Ivoire). Geoderma 56, 119e132. Blanchart, E., Albrecht, A., Alegre, J., Duboisset, A., Gilot, C., Pashanasi, B., Lavelle, P., Brussaard, L., 1999. Effects of earthworms on soil structure and physical properties. In: Lavelle, P., Brussaard, L., Hendrix, P. (Eds.), Earthworm Management in Tropical Agroecosystems. CAB International, pp. 149e171. Bossuyt, H., Six, J., Hendrix, P.F., 2004. Rapid incorporation of carbon from fresh residues into newly formed stable microaggregates within earthworm casts. European Journal of Soil Science 55, 393e399. Bossuyt, H., Six, J., Hendrix, P.F., 2005. Protection of soil carbon by microaggregates within earthworm casts. Soil Biology and Biochemistry 37, 251e258. Bouché, M.B., 1977. Stratégies lombriciennes. Ecological Bulletin 25, 122e132. Bracewell, J.M., Pacey, N., Robertson, G.W., 1987. Organic matter in onshore cretaceous chalks and its variations, investigated by pyrolysis-mass spectrometry. Journal of Analytical and Applied Pyrolysis 10, 199e213.
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