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Do earthworms affect the fractionation of silicon in soil?
Pedobiologia - Journal of Soil Ecology 75 (2019) 1–7
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Do earthworms affect the fractionation of silicon in soil? a,⁎
b
a
a
T a
Anna Georgiadis , Sven Marhan , Adrian Lattacher , Philipp Mäder , Thilo Rennert a b
Fachgebiet Bodenchemie mit Pedologie, Institut für Bodenkunde und Standortslehre, Universität Hohenheim, Emil-Wolff-Str. 27, 70599 Stuttgart, Germany Fachgebiet Bodenbiologie, Institut für Bodenkunde und Standortslehre, Universität Hohenheim, Emil-Wolff-Str. 27, 70599 Stuttgart, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Sequential extraction Silicic acid Octolasion cyaneum Savigny Eisenia andrei Bouché Aggregates
It is known that earthworms can increase the content of water-extractable silicon (Si) in soil, thus contributing to the availability of Si for plants. However, effects of earthworms on other Si fractions in soil, such as adsorbed Si, Si bound to SOM, Si occluded in pedogenic oxides and amorphous silica have not yet been studied. Therefore, we investigated the effects of the endogeic earthworm Octolasion cyaneum Savigny on the fractionation of Si in soils and that of the epigeic earthworm Eisenia andrei Bouché on the release of Si from model substances (quartz, wheat straw, bioopal). We quantified the amounts of Si in different soil fractions and those released from model substances before and after passage through the earthworm gut by sequential Si extraction. The amounts of Si extracted from the earthworms’ casts were generally larger than in the undigested samples. This was especially pronounced for Si bound to soil organic matter (SOM; up to 41%), and for Si in wheat straw (up to 71%) and quartz (up to 1730%). With the soils, the increase in extracted Si was pronounced for the more mobile fractions (Si bound to SOM and occluded in pedogenic oxides) at the expense of amorphous silica. The amounts of mobile and adsorbed Si (plant-available Si) in soil tended to decrease after the passage through the earthworm gut, possibly by occlusion of adsorbents in aggregates formed in the gut. Our results indicate that both mechanical weathering of ingested Si-containing particles and microbial processes, promoting aggregate formation, SOM transformation and mineral solubilization, contribute to the increased release of Si, which induced the redistribution of Si among fractions.
1. Introduction Silicon is an important element for plant development (Epstein, 2001). It enhances plant tolerance to a wide range of biotic (e.g., herbivores and pathogens) and abiotic (e.g., presence of metals, salt and drought) stress factors (Ma et al., 2001; Zhu and Gong, 2014; Tubana and Heckman, 2015). Its availability can greatly improve the yield of crops such as rice (Li et al., 2013), wheat (Gocke et al., 2013) and sugarcane (Savant et al., 1999). The Si content of soils varies from < 10 up to 450 g kg−1 (Sommer et al., 2006). Silicon released by weathering of primary silicates in soil may be leached out or is consumed by the formation of secondary silicates. It may precipitate as amorphous silica, be adsorbed by pedogenic oxides and hydroxides, or be taken up by plants (Sommer et al., 2006). The plants exclusively take up Si as monomeric silicic acid (H4SiO4; pKa1 = 9.8; Haynes, 2014), which is transported into plant organs, where it polymerizes as hydrous amorphous silica or bioopal, so-called phytoliths. During the decomposition of plants, bioopal Si is returned into soil (Kaufman et al., 1981) so that a large proportion of amorphous silica in most soils is comprised by bioopal (Drees et al.,
⁎
1989). Earthworms play an important role in the improvement of soil physical conditions and plant growth (Winding et al., 1997; Scheu, 2003). They regulate the soil structure by creating burrows, which facilitate water and gas transport, by mixing soil minerals with organic material (bioturbation) and by creating aggregates (Winding et al., 1997). Earthworms accelerate the degradation of soil organic matter (SOM) by increasing its available surface area by comminution and fostering soil microbial activity (Blouin et al., 2013). Earthworms induce both chemical and physical weathering of minerals, for instance by promoting the transformation of clay minerals and mechanical stress (Suzuki et al., 2003; Carpenter et al., 2007). Small mineral particles in the earthworm gut contribute to grinding of plant material as well as physical weathering of minerals in the muscular gizzard (Schulmann and Tiunov, 1999; Marhan and Scheu, 2005). Bacteria living in the earthworm gut enhance the solubility of silicates (Hu et al., 2018). The authors carried out experiments with earthworm-gut bacteria and demonstrated that inoculation of potting soil with these bacteria promoted growth of maize plants. Bityutskii et al. (2016) reported that earthworms increase the content of water-extractable (thus plant-
Corresponding author. E-mail address: [email protected] (A. Georgiadis).
https://doi.org/10.1016/j.pedobi.2019.05.001 Received 20 December 2018; Received in revised form 2 May 2019; Accepted 7 May 2019 0031-4056/ © 2019 Elsevier GmbH. All rights reserved.
Pedobiologia - Journal of Soil Ecology 75 (2019) 1–7
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available) Si in soil after passage through the earthworm gut. However, up to now the source of the additional plant-available Si provided by actions of earthworms is unknown. Other Si fractions such as adsorbed Si, Si bound to SOM, Si occluded in pedogenic oxides and amorphous silica are potential sources of plant-available Si after mobilization from these fractions and its redistribution induced in the earthworm gut. Thus, these fractions may act as important sources for dissolved Si that can be taken up by plants. Therefore, the knowledge about the potential of earthworms to increase Si availability can be important for sustainable soil management. The aim of this study was to clarify the effects of earthworms on the release of Si from soil constituents and on the distribution of Si among operationally defined fractions in soil. We hypothesize that chemical, biological and physical weathering of Si-containing minerals together with transformation of SOM during the passage through the earthworm gut lead to increasing Si solubilization in soil and to a redistribution of Si among fractions. We expect an increase in the amounts of Si in more mobile fractions at the expense of Si in less mobile fractions after the passage of soil through the earthworm gut. For a quantification of Si among fractions, we applied a sequential Si extraction procedure (Georgiadis et al., 2013) on selected soil samples to quantify Si in soil in mobile and adsorbed (plant-available) form, Si bound to SOM, Si occluded in pedogenic oxides and amorphous silica before and after the passage of soil through the earthworm gut. In additional independent experiments, we checked the effects of earthworms on the release of Si from models of Si-containing soil constituents. These model substances comprised quartz, bioopal and wheat straw. We used quartz as a model of a crystalline Si form, bioopal as a model of amorphous Si and wheat straw as model of Si-containing plant material in soil. Using defined model substances may help to explain the findings from the experiments with soil and thus to differentiate the superimposing processes that occur during the (re-) distribution of Si in a complex mixture like soil. We tested the effects of endogeic earthworms on the distribution of Si among fractions on soil samples from three sites with different land use, representing different earthworm habitats. We used endogeic earthworms, as they are abundant in the studied areas. As geophagous species, they contribute to silicate weathering and Si mobilization in soil (Bityutskii et al., 2016; Hu et al., 2018). In the approach with model substances, we used epigeic earthworms, as they strongly affect transformation of organic matter and may thus mobilize Si (Sims and Gerard, 1999; Bityutskii et al., 2016).
Table 1 Selected properties of soil samples used for the experiments with Octolasion cyaneum Savigny (experiment a). Soils
Horizon
Corga Nb [g kg−1]
Sand
Silt %
Clay
pHc
pHc (casts)
Stagnic Luvisol Haplic Luvisol Haplic Umbrisol
Ap Ah Ap
9.0 46.7 36.2
3 2 7
72 76 64
25 22 29
6.0 3.9 5.0
6.2 3.9 5.0
a b c
1.1 3.6 3.1
Organic carbon. Total nitrogen. Measured in CaCl2 solution at a solid-to-solution ratio of 1:10.
The model substances (Table 2) were mixed with peat that was used as an experimental matrix for feeding earthworms to ensure their survival in the experiments. Pure peat served as control. 2.2. Experimental setup We carried out two sets of experiments - (a) with soil samples and the endogeic earthworm species Octolasion cyaneum Savigny that is geophagous and lives mostly in the upper mineral soil, and (b) with model substances and the epigeic species Eisenia andrei Bouché that thrives in compost and manure and feeds almost exclusively on organic matter (Sims and Gerard, 1999). O. cyaneum were collected by the Thielemann octet method and hand sorting from a garden near Stuttgart (Germany). E. andrei species were collected by hand sorting from compost. Prior to the experiments, O. cyaneum species were kept 72 h in fresh soil for adaptation. E. andrei were kept for one week in peat for adaptation and then depurated in moistened Petri dishes for 24 h for defecation, before being added to the mixtures of model substances with peat. For experiment (a), we placed about 15 g of each soil sample in a Petri dish (in total, six Petri dishes per sample) and adjusted the gravimetric water content to 36–38%. Subsequently, one O. cyaneum specimen was placed into each of three Petri dishes per sample. A further three Petri dishes were left without earthworms as a control (i.e., three replicates with earthworms and three control replicates for each sample; in total 18 Petri dishes). The earthworm casts after single ingestion, produced after two days were collected, dried at 30 °C and stored in plastic vessels. For experiment (b), we mixed 12 g of wheat straw (milled to powder), 7 g of quartz and 5 g of bioopal separately with peat (5–12 g, depending on the model substance; Table 3). We placed each mixture in a Petri dish (in total four Petri dishes per sample). A further four Petri dishes contained only peat (control). Subsequently, we placed E. andrei in two Petri dishes per sample (20 specimens per Petri dish). A further two Petri dishes were left without earthworms (two replicates with and two replicates without earthworms for each mixture and pure peat; in total 16 Petri dishes). The water content of the mixtures was adjusted based on the earthworms’ activity in the Petri dishes to achieve optimum conditions for their adaptation (Table 3). After 24 h, we rinsed the earthworms with water and placed them in a separate Petri dish until the earthworms produced casts. Then, we dried the casts at 30 °C and stored them in plastic vessels. We did not use glassware to minimize Si contamination.
2. Materials and methods 2.1. Soils and model substances We sampled the A horizons from soil profiles on three study sites that represent typical sites of arable land, grassland and forest of southwest Germany. Three replicates per site (each 500 g) were pooled to one composite sample per site. The soils comprised a Stagnic Luvisol (SL; WRB classification (IUSS Working Group WRB, 2015)) developed from loess (arable land), a Haplic Umbrisol (HU) developed from colluvial loam (grassland, formerly arable land) and a forested Haplic Luvisol (HL) developed from loess. The soil samples were passed through a 2-mm sieve and stored at 5 °C. Particle-size distribution was determined by wet sieving (sand) and sedimentation (silt and clay). For consistency, we determined the pH of both, the soil samples and the earthworm casts, with a glass electrode in 0.01 M CaCl2 at a solid-to-solution ratio (SSR) of 1:10, as the amount of earthworm casts obtained in experiments was scarce. Total carbon (C) and nitrogen (N) contents of the soils were determined by elemental analysis (dry combustion) (Vario MACRO, Elementar, Hanau, Germany). The soil samples were free of carbonates, thus the contents of soil organic C equaled those of total C. Soil properties are summarized in Table 1.
2.3. Analyses After drying (35 °C) the soil samples, model substances with peat and pure peat as well as the respective earthworm casts, we applied a sequential extraction scheme (Georgiadis et al., 2013) to quantify Si in different fractions, which included: 1) Mobile Si (Sil), extracted by 0.01 M CaCl2, SSR (solid-to-solution ratio) 1:10, 2
Pedobiologia - Journal of Soil Ecology 75 (2019) 1–7
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Table 2 Model substances used for the experiments with Eisenia andrei Bouché (experiment b). Substance
Characteristics
pHa
Corg
Peat Quartz Bioopal Wheat straw
< 2 mm, culture substrate TKS® 1 H 32, average particle size 320 μm from Equisetum arvense harvested from arable land near Stuttgart Triticum aestivum, harvested from arable land
6.2 5.6 4.0 6.1
480 n.d. 16.2 427
N [g kg−1] 9.98 n.d. 0.29 4.86
Sitotalb
Source
0.14 n.d. n.d. 6.5
Floragard, Oldenburg, Germany Quarzwerke Frechen, Germany obtained after Bartoli and Wilding (1980) –
n.d. not determined. a Measured in H2O at a solid-to-solution ratio of 1:25. b Quantified after Georgiadis et al. (2015).
2) Adsorbed Si (Siad), extracted by 0.01 M acetic acid, SSR 1:10, 3) Si bound to SOM (Siorg), released by concentrated H2O2 at 85 °C, SSR 1:30, 4) Si occluded in pedogenic oxides and hydroxides (Siocc), released by 0.2 M NH4-oxalate under UV radiation, SSR 1:50 and 5) Si in amorphous silica (Siam), extracted by 0.2 M NaOH after 168 h, SSR 1:400.
2.4. Statistics
For experiment (b), we restricted the extraction to steps 1–3 and 5 (SSR 1:20 in steps 1 and 2, and 1:60 in step 3), as we did not use any oxide as model substance. Samples with bioopal were not subjected to step 5, since bioopal is completely dissolved in this step, even without passage through the earthworm gut (Georgiadis et al., 2015). The SSRs were adapted for optimal Si extraction. All extracts were centrifuged (1400 g) until the supernatant was clear and then filtered through paper filters (1–2 μm). Silicon in the extracts was analyzed by microwave plasma-atomic emission spectrometry (4200 MP-AES, Agilent, Waldbronn, Germany). MP-AES, like ICP-AES and -OES (inductively coupled plasma optical emission spectrometry), bases upon the release of element-specific electromagnetic radiation from excited atoms. In an MP-AES system, N2 fuels a microwave plasma utilized for excitation. We analyzed Si using its most sensitive atomic line with negligible spectral interferences at 288.158 nm. Atomic lines are less susceptible to matrix effects (Karlsson et al., 2015). Quality control included calibration and measuring samples with known concentration for each series of measurements in the respective matrix (extractant solution). The precision in all Si analyses was < 5% (relative standard deviation). The detection limit for Si in 0.01 M CaCl2 (the extractant with the lowest Si concentrations measured) was 20 μg l−1. The amounts of extracted Si were related to the material dried at 30 °C. The amounts of Si extracted from the model substances (experiment (b)) were corrected for the Si amounts determined in pure peat obtained in identical experiments. About 5–10 particles from each experiment (b) were mounted on an Al sample holder with double-sided adhesive and conductive carbon tape, sputtered with a thin layer of Au/Pd alloy. We analyzed them by scanning-electron microscopy (LEO 420, LEO Electron Microscopy, Cambridge, UK) in secondary-electron mode to visualize particles before and after their passage through the earthworm gut.
3. Results
We used SigmaPlot 11.0 (Systat Software Inc., San Jose, USA) for statistical analyses. We used the Spearman rank correlation coefficient (rs) to correlate soil data with non-linear distribution. Levels of statistical significance were *** P ≤ 0.001, ** 0.001 < P ≤ 0.01, * 0.01 < P ≤ 0.05 and not significant when P > 0.05.
3.1. Experiment (a) - Effects of endogeic earthworms on the fractionation of Si in soil Relative to the earthworm-free soil samples, the sum of Si extracted from the earthworms’ casts in the five extraction steps increased by 10% and 28% (SL and HL, respectively), but decreased negligibly by 2.5% (HU, Fig. 1a). The passage through the earthworm gut hardly affected the contents of mobile Sil and adsorbed Siad in all soil samples. The amounts of Sil and Siad extracted from the casts increased by 4% and 2% (SL), and decreased by 4.5% and 5% (HU) and by 5% and 4% (HL), respectively, relative to the earthworm-free samples (Fig. S1). Overall, Sil and Siad contents were smallest among all Si fractions. The Sil contents were considerably lower for the SL samples (7.4 and 7.7 mg kg−1 for the earthworm-free samples and casts, respectively; Table S1) than for the HU (11.8 and 11.2 mg kg−1 for the earthworm-free samples and casts, respectively) and HL samples (12.5 and 11.9 mg kg−1 for the earthworm-free samples and casts, respectively). The Siad contents were significantly larger in SL samples (98 and 100 mg kg−1 for the earthworm-free samples and casts, respectively) than in the HU (29 and 27 mg kg−1 for the earthworm-free samples and casts, respectively) and HL samples (30 and 29 mg kg−1 for the earthworm-free samples and casts, respectively). The Sil contents were positively correlated with c (H+) (rs = 0.8***) and the contents of Corg (rs = 0.8***) and N (rs = 0.8***). The Siad contents of the earthworm-free soil were negatively correlated with c(H+) (rs = -0.9***) and the Sil (rs = -0.8***), Corg (rs = -0.9***) and N (rs = -0.9***) contents. The Siad contents of the casts were not correlated with any other soil parameter. The contents of Si bound to SOM were positively affected by O. cyaneum. Extraction of Siorg from the casts increased by 7%, 19% and 41% (SL, HU and HL, respectively, Fig. 1b). The Siorg contents of all samples were strongly negatively correlated with the clay contents (rs = -0.9***).
Table 3 Properties of model substances and experimental conditions used in experiments with Eisenia andrei Bouché. Substance
Mass ratio of model substance to peat
Quartz mixed with peat Bioopal mixed with peat Wheat straw mixed with peat Pure peat
1:1 1:1 1:2 –
a
Mass per Petri dish [g] 14 10 18 10
Measured in H2O at a solid-to-solution ratio of 1:25. 3
Water content [wt%] 125 300 300 300
pHa
pHa (casts)
6.5 5.9 6.4 6.3
7.2 7.3 7.7 7.5
Pedobiologia - Journal of Soil Ecology 75 (2019) 1–7
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Fig. 1. Experiment (a): Contents of Si extracted from soil before (white) and after (grey) the passage through the gut of Octolasion cyaneum Savigny according to sequential extraction (SL, Stagnic Luvisol; HU, Haplic Umbrisol; HL, Haplic Luvisol): a) total Si extracted; b) Si bound to soil organic matter; c) Si from amorphous silica and d) Si occluded in pedogenic oxides. Data are expressed as arithmetical means ± standard deviation (n = 3).
extraction from bioopal were distinctly smaller, with a decrease by 16% (first step), and increases by 8% (second step) and 95% (third step), respectively (Fig. 2c). The smallest total contents of Si extracted were found for the quartz samples (0-0.14 mg g−1; Table S2) and the largest (1.2–5.4 mg g−1) for the bioopal samples. Treatment with H2O2 released the largest amounts of Si from all samples. Extraction with 0.2 M NaOH (fifth step) released no Si from quartz in both experiments, and relatively small amounts from wheat straw (0.91 and 0.93 mg g−1 for the undigested samples and casts, respectively). The largest changes in the distribution of Si among forms were found for quartz. The casts exhibited an increase of Si shares by six and 34 percentage points after extraction in the first and second step, respectively, and a decrease by 40 percentage points after extraction with H2O2 in the third step. The Si shares of the casts of the wheat-straw samples increased by three percentage points in the first and second extraction steps and decreased by three percentage points in the third and fifth extraction steps, respectively. The redistribution of Si shares in the samples with bioopal was negligible. Again, it should be considered that the results of this experiment are also based on single ingestion of model substances by earthworms. The passage of all model substance mixed with peat through the gut of E. andrei increased the pH by 0.7 (quartz), 1.4 (bioopal), 1.3 (wheat straw) and 1.2 (pure peat).
The contents of Siocc extracted from the casts were larger than extracted from the earthworm-free samples with increases of 8%, 6% and 38% (SL, HU and HL, respectively, Fig. 1d). In contrast to Siorg, the Siocc contents of all samples were positively correlated with clay contents (rs = 0.7*). The contents of Si from amorphous silica were larger for the casts (increase of 13% and 29%) of the SL and HL samples and lower for the casts of the HU samples (decrease of 5%), relative to the earthwormfree samples. Silicon in amorphous silica constituted the largest Si fraction, making up 1.8–2.6 mg g−1 (Fig. 1c). The Siam contents of all samples were positively correlated with clay contents (rs = 0.9***) and negatively with the Siorg contents (rs = -0.8***/** earthworm-free/ cast). The largest changes in the distribution of Si among the fractions were found for the HL samples after their passage through the earthworms’ guts. Relative to the earthworm-free treatment, the share of Siorg in the total extractable Si increased by 2.1 percentage points, while that of Siam and Siad decreased by 1.5 and 0.7 percentage points, respectively. In the casts of the HU samples, the shares of Siorg and Siocc increased by 1.3 and 1.1 percentage points, respectively. Changes in the distribution of Si among fractions after passage through the earthworm gut were negligible for the SL samples. The pH of the SL samples (6.0–6.2) was slightly increased in casts of O. cyaneum, while pH values of other samples were not changed after the passage through the earthworm gut relative to the earthworm-free soils (Table 1). However, we have to keep in mind that all these results are based on single ingestion of soil by earthworms and might become more pronounced when repeated, on the time scale of years.
4. Discussion 4.1. Effects of earthworms on contents of plant-available Si and Si bound to SOM
3.2. Experiment (b) - Effects of epigeic earthworms on Si extraction from model substances in a peat matrix
We detected an increase in the contents of Si extracted from soils and model substances after the passage through the earthworm gut. This increase was especially pronounced for the HL samples, wheat straw and quartz. Silicon bound to SOM was the major contributor to the additional extractable Si from soils. The largest increase in the contents of Siorg (by 41%) was detected for the HL samples with the largest organic C content among the soils under study. An increase in the contents of Siorg in the casts of all soil samples and an increase in total extractable Si in the casts of the wheat straw indicate that earthworms transformed SOM (Drake and Horn, 2007). Transformation and degradation of SOM during the passage of the earthworm gut induce the release of Si that is bound to SOM and make it more easily
Unlike for the bioopal samples, the total amounts of Si extracted from the model substances increased after passage through the earthworm gut, by 257% for quartz and by 20% for wheat straw, relative to undigested samples (Fig. 2a). Specifically, Si extracted from the casts of the quartz samples increased by 764% in the first step (extracted by CaCl2), 1730% in the second step (acetic acid) and 95% in the third step (H2O2), relative to the undigested samples (Fig. 2b). E. andrei had smaller effects on Si extraction from wheat-straw samples with increases in Si extraction by 71%, 38%, 13% and 3% in the first, second, third and fifth step, respectively. The earthworm-induced effects on Si 4
Pedobiologia - Journal of Soil Ecology 75 (2019) 1–7
A. Georgiadis, et al.
Fig. 2. Experiment (b): Contents of Si extracted from model substances mixed with peat before (white) and after (grey) the passage through the gut of Eisenia andrei Bouché according to sequential extraction: a) total Si extracted; b) quartz; c) bioopal and d) wheat straw. Step 1: extracted by 0.01 M CaCl2; Step 2: extracted by 0.01 M acetic acid; Step 3: released by concentrated H2O2; Step 5: extracted with 0.2 M NaOH. Pure peat was used as control. Data are expressed as arithmetical means ± standard deviation (n = 2). Please note the different scales on the y-axes.
aggregates (Emerson et al., 1986). We detected such aggregates in the earthworms’ casts of quartz (Fig. 3a) mixed with peat as patches of peat particles adhered to quartz grains (Fig. 3b). Additionally, during the passage through the earthworm gut, minerals may be mechanically altered. Suzuki et al. (2003) detected finer and rounded feldspar and quartz grains after the passage of earthworms. Extractability of Si from such mineral grains and from the scraped material should increase because of an increase in the surface area, relative to undigested mineral grains. Thus, mechanical weathering of quartz particles, together with an increase in solubilization induced by microbial activity, may explain the large increase in the amounts of Si extracted from the casts of the quartz samples in our study. Therefore, earthworms contribute to mobilization of plantavailable Si from quartz and very likely other Si-containing minerals. However, the contribution of plant-available Si from quartz in soil is presumably small. Sommer et al. (2002) roughly estimated the production of cast by earthworms to 5 kg m−2 a-1 for soil in the area under study. Assuming that quartz dominates the sand fraction (accounting for 2–7% of the total soil; Table 1), 15–50 mg plant-available Si m−2 a-1 is provided. This amount of Si, provided by quartz, corresponds to 5–8% of plant-available Si (as the sum of Sil and Siad) that is present in the earthworms’ casts produced in the experiments with soil. Apart from the general low susceptibility of quartz to physical and chemical weathering, the small contribution of quartz to plant-available Si resulted from single ingestion of soil by earthworms, which could increase after repeated ingestion on a larger time scale.
extractable (Bityutskii et al., 2016). Furthermore, Si released from the earthworm casts of the soil samples by H2O2 may partially represent mobile and adsorbed Si protected within aggregates and thus not accessible for extraction in the first two extraction steps. This was indicated by decreased contents of Sil and Siad in the casts of HU and HL. Earthworms produce compact aggregates during digestion (Jouquet et al., 2008). We detected such aggregates in the casts of model substances mixed with peat (Fig. 3). The aggregates may have been formed by glue produced by gut bacteria or by reinforcement by fibrous plant residues (Lee and Foster, 1991). Oxidizing the aggregating SOM by H2O2 may have led to the dispersion of aggregates so that Sil and Siad previously present in the aggregates may have been released into solution. The strong negative correlation between the contents of Sil and Siad and the strong positive correlation of the contents of Sil with the Corg and N contents in our study point to an association of these Si species with SOM. The partly negative to negligible effect of O. cyaneum on the extraction of mobile and adsorbed Si is in contrast to the findings by Bityutskii et al. (2016). They detected an increase in the contents of water-extractable Si after the passage of soil through the guts of a endogeic earthworm species, Aporrectodea caliginosa. Like in our approach, Bityutskii et al. (2016) determined the Si contents in the earthworm casts after single ingestion of soil. Moreover, the Si firstly released from Si-containing minerals (such as silicates or amorphous silica) and then adsorbed on SOM in the earthworms’ guts, may have contributed to the enhanced contents of Siorg in the earthworms’ casts. Silicon transferred to SOM may then be released by oxidation of SOM by H2O2 treatment. The increase in the Siorg shares in the casts of HL and HU samples was mostly at the expense of Si from amorphous silica. This is also indicated by a close negative correlation between the contents of Siam and Siorg.
4.3. Effects of earthworms on the contents of Si in amorphous silica Earthworms hardly affected the solubilization of Si from bioopal. The solubility of amorphous silica increases with increasing pH (Fraysse et al., 2009). Usually, the pH of earthworm gut is neutral to slightly alkaline (6.4–7.7) so that homeostasis occurs (Drake and Horn, 2007). E. andrei increased the pH of the casts of all model substances (Table 3). However, slightly less Si was extracted from the casts of bioopal mixed with peat in the first extraction step. This may be explained by a protective effect of the peat: After passage of the bioopal-peat mixture through the earthworm gut, coarse bioopal-peat aggregates have formed (Fig. 3c, d). The small bioopal particles are embedded into coarser peat structures and are thus less available for extraction. These aggregates were destroyed during the first extraction step by shaking the samples, resulting in slightly larger Si release in the second
4.2. Effects of earthworms on the release of Si from soil minerals Microorganisms living in the earthworm gut may promote weathering of Si-containing minerals and increase Si extractability (Hu et al., 2018). Microbes can colonize mineral surfaces. Protons, extracellular enzymes, metabolic by-products, organic acids and chelators, produced by microbes may induce dissolution of minerals in the earthworm gut (Bennett et al., 2001; Uroz et al., 2009; Hu et al., 2018). These microbial products can interact with the surfaces of mineral particles and contribute to the formation of organo-mineral complexes and 5
Pedobiologia - Journal of Soil Ecology 75 (2019) 1–7
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Fig. 3. Secondary electron images of quartz (a, b), bioopal (c, d) and wheat straw (e, f) mixed with peat before (a, c, e) and after (b, d, f) the passage through the gut of Eisenia andrei Bouché. The white scale bar represents 20 μm. Q, quartz; B, bioopal; W, wheat straw; P peat.
4.4. Effects of earthworms on the contents of Si occluded in pedogenic oxides
extraction step. This is supported by the changes in Si shares after passage of bioopal through the earthworm gut, relative to the undigested bioopal: The share of Si in the cast decreased after the first extraction step and increased after the second and third. Nonetheless, the effect of earthworms on Si extraction from bioopal was the smallest among all model substances and may be considered negligible for the mobilization of Si. In the experiments with soils, the pH values of the casts did not change. However, the amounts of Siam extracted from the casts of SL and HL samples were distinctly larger than extracted from the earthworm-free samples. We detected the largest effect of earthworms on Siam extraction from the HL samples of the forested site (increase of Siam in the casts by 29%). Usually, bioopal is the most abundant form of amorphous silica in topsoil (Georgiadis et al., 2014), mostly as phytolith. In contrast to the agricultural sites, bioopal particles in the forested soil were not crushed mechanically by soil cultivation, e.g. by ploughing. Thus, these relatively large bioopal particles can be crushed into smaller particles by muscular contractions of the earthworms’ gizzard (Edwards and Bohlen, 1996). Subsequently, these particles became more easily extractable by NaOH. Thus, it is likely that the mechanical stress during the passage of the earthworm gut is more important for bioopal-Si mobilization than the change in pH in the earthworm gut. However, the effect of the gut milieu may become more pronounced after repeated passage of bioopal through the earthworm gut.
Bacteria of the earthworm gut may participate in redox reactions. Pseudomonas and Bacillus species have been detected in the gut of endogeic earthworms (Drake and Horn, 2007; Hu et al., 2018). They can reduce Mn(IV) and Fe(III) (Sundaram et al., 2012) and thus may induce partial reductive dissolution of Fe/Mn oxides, which are important sorbents of Si species (Georgiadis et al., 2013, 2017), and of Fe(Mn)containing silicates, which would induce the release of bound Si into the earthworm gut. After excretion of the casts and exposure to the atmosphere, Fe and Mn oxides may subsequently precipitate and retain previously released Si by adsorption, (co-)precipitation or occlusion. Silicon from other sources released in the earthworm gut (e.g., Si from amorphous silica) may be involved in these sorption processes as well. This may help to explain the higher amounts of Siocc extracted from the earthworm casts (increase up to 38%) as well as the changes in the distribution of Si shares in the casts of HU and HL samples with increase in Siocc and decrease in Siam. 5. Conclusions Our study demonstrates that earthworms (O. cyaneum and E. andrei) affect the distribution of Si among fractions by the passage of soil through their gut, even after a single ingestion. They increased the 6
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solubilization of Si in soil, inducing the redistribution of Si among the fractions. Earthworms induced the formation of aggregates, possibly occluding plant-available Si. On the other hand, they induced a slight mobilization of plant-available Si from quartz. However, the proportion of Si extractable from quartz in soil casts was low, amounting to 5–8% of the whole plant-available Si in soil casts. The major contributor to the additional extractable Si from the casts was Si bound to SOM, indicating transformation of SOM by earthworms. The Si occluded in oxides and hydroxides as well as amorphous silica also contributed to the increase of extractable Si from casts. The sum of extractable Si from all analyzed fractions in the casts may be considered as potentially plant-available Si. Thus, earthworm casts may act as an important source for this kind of Si in soil, with larger contents than the surrounding soil. The Si may become available for plants after destruction of the aggregates. However, stability of such aggregates as well as the Si release in the soil solution after their destruction has not yet been studied. The role of such aggregates for the long-term Si release and retention is neither clarified. On the one hand, Si release from mineral particles after their passage through the earthworm gut increases due to mechanical stress or microbially induced weathering. On the other hand, organic matter, covering or embedding these particles can oppositely pose as sorbent for previously released Si. These open questions may be answered in long-term experiments that offer the possibility of repeated ingestion of soil by earthworms.
reactivity of plant phytoliths in aqueous solutions. Chem. Geol. 258, 197–206. Georgiadis, A., Sauer, D., Herrmann, L., Breuer, J., Zarei, M., Stahr, K., 2013. Development of a method for sequential Si extraction from soils. Geoderma 209–210, 251–256. Georgiadis, A., Sauer, D., Herrmann, L., Breuer, J., Zarei, M., Stahr, K., 2014. Testing a new method for sequential Si-extraction on soils of temperate-humid climate. Soil Res. 52, 645–657. Georgiadis, A., Sauer, D., Breuer, J., Herrmann, L., Rennert, T., Stahr, K., 2015. Optimising the extraction of amorphous silica by NaOH from soils of temperatehumid climate. Soil Res. 53, 392–400. Georgiadis, A., Rinklebe, J., Straubinger, M., Rennert, T., 2017. Silicon fractionation in Mollic Fluvisols along the Central Elbe River, Germany. Catena 153, 100–105. Gocke, M., Liang, W., Sommer, M., Kuzyakov, Y., 2013. Silicon uptake by wheat: effects of Si pools and pH. J. Plant Nutr. Soil Sci. 176, 551–560. Haynes, R.J., 2014. A contemporary overview of silicon availability in agricultural soils. J. Plant Nutr. Soil Sci. 177, 831–844. Hu, L., Xia, M., Lin, X., Xu, C., Li, W., Wang, J., Zeng, R., Song, Y., 2018. Earthworm gut bacteria increase silicon bioavailability and acquisition by maize. Soil Biol. Biochem. 125, 215–221. IUSS Working Group WRB, 2015. World Reference Base for Soil Resources 2014, Update 2015. International Soil Classification System for Naming Soils and Creating Legends for Soil Maps. World Soil Resources Reports No. 106. FAO, Rome. Jouquet, P., Bottinelli, N., Podwojewski, P., Hallaire, V., Duc, T.T., 2008. Chemical and physical properties of earthworm casts as compared to bulk soil under a range of different land-use systems in Vietnam. Geoderma 146, 231–238. Karlsson, S., Sjöberg, V., Ogar, A., 2015. Comparison of MP AES and ICP-MS for analysis of principal and selected trace elements in nitric acid digests of sunflower (Helianthus annuus). Talanta 135, 124–132. Kaufman, P.B., Dayanandan, P., Takeoka, Y., Bigelow, W.C., Jones, J.D., Iler, R., 1981. Silica in shoots of higher plants. In: Simpson, T.L., Volcani, B.E. (Eds.), Silicon and Siliceous Structures in Biological Systems. Springer, New York, pp. 409–449. Lee, K.E., Foster, R.C., 1991. Soil fauna and soil structure. Aust. J. Soil Res. 29, 475–775. Li, Z.M., Song, Z.L., Parr, J.F., Wang, H.L., 2013. Occluded C in rice phytoliths: implications to biogeochemical carbon sequestration. Plant Soil 370, 615–623. Ma, J.F., Miyake, Y., Takahashi, E., 2001. Silicon as a beneficial element for crop plants. In: Datnoff, L.E., Snyder, G.H., Korndörger, G.H. (Eds.), Silicon in Agriculture. Elsevier Science B.V., Amsterdam, pp. 17–39. Marhan, S., Scheu, S., 2005. Effects of sand and litter availability on organic matter decomposition in soil and in casts of Lumbricus terrestris L. Geoderma 128, 155–166. Savant, N.K., Korndörfer, G.H., Datnoff, L.E., Snyder, G.H., 1999. Silicon nutrition and sugarcane production: A review. J. Plant Nutr. 22, 1853–1903. Scheu, S., 2003. Effects of earthworms on plant growth: patterns and perspectives. Pedobiologia 47, 846–856. Schulmann, O.P., Tiunov, A.V., 1999. Leaf litter fragmentation by the earthworm Lumbricus terrestris L. Pedobiologia 43, 453–458. Sims, R.W., Gerard, B.M., 1999. Earthworms. FSC Publications, London. Sommer, M., Ehrmann, O., Friedel, J.K., Martin, K., Vollmer, T., Turian, G., 2002. Böden als Lebensraum für Organismen - Regenwürmer, Gehäuselandschnecken und Bodenmikroorganismen in Wäldern Baden-Württembergs. Hohenheimer Bodenkundliche Hefte 63. Universität Hohenheim, Stuttgart. Sommer, M., Kaczorek, D., Kuzyakov, Y., Breuer, J., 2006. Silicon pools and fluxes in soils and landscapes - a review. J. Plant Nutr. Soil Sci. 169, 310–329. Sundaram, P.A., Augustine, R., Kannan, M., 2012. Extracellular biosynthesis of iron oxide nanoparticles by Bacillus subtilis strains isolated from rhizosphere soil. Biotechnol. Bioprocess Eng. 17, 835–840. Suzuki, Y., Matsubara, T., Hoshino, M., 2003. Breakdown of mineral grains by earthworms and beetle larvae. Geoderma 112, 131–142. Tubana, B.S., Heckman, J.R., 2015. Silicon in soils and plants. In: Rodrigues, F.A., Datnoff, L.E. (Eds.), Silicon and Plant Diseases. Springer, Cham, pp. 7–51. Uroz, S., Calvaruso, C., Turpault, M.-P., Frey-Klett, P., 2009. Mineral weathering by bacteria: ecology, actors and mechanisms. Trends Microbiol. 17, 378–387. Winding, A., Rønn, R., Hendriksen, N.B., 1997. Bacteria and protozoa in soil microhabitats as affected by earthworms. Biol. Fertil. Soils 24, 133–140. Zhu, Y., Gong, H., 2014. Beneficial effects of silicon on salt and drought tolerance in plants. Agron. Sustain. Dev. 34, 455–472.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.pedobi.2019.05.001. References Bartoli, F., Wilding, L.P., 1980. Dissolution of biogenic opal as a function of its physical and chemical properties. Soil Sci. Soc. Am. J. 44, 873–878. Bennett, P.C., Rogers, J.R., Choi, W.J., Hiebert, F.K., 2001. Silicates, silicate weathering, and microbial ecology. Geomicrobiol. J. 18, 3–19. Bityutskii, N., Kaidun, P., Yakkonen, K., 2016. Earthworms can increase mobility and bioavailability of silicon in soil. Soil Biol. Biochem. 99, 47–53. Blouin, N., Hodson, M.E., Delgado, E.A., Baker, G., Brussard, L., Butt, K.R., Dai, J., Dendooven, L., Peres, G., Tondoh, J.E., Cluzeau, D., Brun, J.-J., 2013. A review of earthworm impact on soil function and ecosystem. Eur. J. Soil Sci. 64, 161–182. Carpenter, D., Hodson, M.E., Eggleton, P., Kirk, C., 2007. Earthworm induced mineral weathering: Preliminary results. Eur. J. Soil Biol. 43, 176–183. Drake, H.L., Horn, M.A., 2007. As the worm turns: the earthworm gut as a transient habitat for soil microbial biomes. Annu. Rev. Microbiol. 61, 169–189. Drees, L.R., Wilding, L.P., Smeck, N.E., Senkayi, A.L., 1989. Silica in soils: quartz and disordered silica polymorphs. In: Dixon, J.B., Weed, S.B. (Eds.), Minerals in Soil Environments, second ed. Soil Sci. Soc. Am., Madison, Wisconsin, USA, pp. 913–974. Edwards, C.A., Bohlen, P.J., 1996. Biology and Ecology of Earthworms, 3rd ed. Chapman and Hall, London. Emerson, W.W., Foster, R.C., Oades, J.M., 1986. In: Huang, P.M., Schnitzer, M. (Eds.), Interactions of Soil Minerals with Natural Organics and Microbes. SSSA Spec. Pub. 17, Madison, Wisconsin, pp. 521–548. Epstein, E., 2001. Silicon in plants: facts vs. concepts. In: Datnoff, L.E., Snyder, G.H., Korndörger, G.H. (Eds.), Silicon in Agriculture. Elsevier Science B.V., Amsterdam, pp. 1–15. Fraysse, F., Pokrovsky, O.S., Schott, J., Meuner, J.-D., 2009. Surface chemistry and
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Pedobiologia - Journal of Soil Ecology journal homepage: www.elsevier.com/locate/pedobi
Do earthworms affect the fractionation of silicon in soil? a,⁎
b
a
a
T a
Anna Georgiadis , Sven Marhan , Adrian Lattacher , Philipp Mäder , Thilo Rennert a b
Fachgebiet Bodenchemie mit Pedologie, Institut für Bodenkunde und Standortslehre, Universität Hohenheim, Emil-Wolff-Str. 27, 70599 Stuttgart, Germany Fachgebiet Bodenbiologie, Institut für Bodenkunde und Standortslehre, Universität Hohenheim, Emil-Wolff-Str. 27, 70599 Stuttgart, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Sequential extraction Silicic acid Octolasion cyaneum Savigny Eisenia andrei Bouché Aggregates
It is known that earthworms can increase the content of water-extractable silicon (Si) in soil, thus contributing to the availability of Si for plants. However, effects of earthworms on other Si fractions in soil, such as adsorbed Si, Si bound to SOM, Si occluded in pedogenic oxides and amorphous silica have not yet been studied. Therefore, we investigated the effects of the endogeic earthworm Octolasion cyaneum Savigny on the fractionation of Si in soils and that of the epigeic earthworm Eisenia andrei Bouché on the release of Si from model substances (quartz, wheat straw, bioopal). We quantified the amounts of Si in different soil fractions and those released from model substances before and after passage through the earthworm gut by sequential Si extraction. The amounts of Si extracted from the earthworms’ casts were generally larger than in the undigested samples. This was especially pronounced for Si bound to soil organic matter (SOM; up to 41%), and for Si in wheat straw (up to 71%) and quartz (up to 1730%). With the soils, the increase in extracted Si was pronounced for the more mobile fractions (Si bound to SOM and occluded in pedogenic oxides) at the expense of amorphous silica. The amounts of mobile and adsorbed Si (plant-available Si) in soil tended to decrease after the passage through the earthworm gut, possibly by occlusion of adsorbents in aggregates formed in the gut. Our results indicate that both mechanical weathering of ingested Si-containing particles and microbial processes, promoting aggregate formation, SOM transformation and mineral solubilization, contribute to the increased release of Si, which induced the redistribution of Si among fractions.
1. Introduction Silicon is an important element for plant development (Epstein, 2001). It enhances plant tolerance to a wide range of biotic (e.g., herbivores and pathogens) and abiotic (e.g., presence of metals, salt and drought) stress factors (Ma et al., 2001; Zhu and Gong, 2014; Tubana and Heckman, 2015). Its availability can greatly improve the yield of crops such as rice (Li et al., 2013), wheat (Gocke et al., 2013) and sugarcane (Savant et al., 1999). The Si content of soils varies from < 10 up to 450 g kg−1 (Sommer et al., 2006). Silicon released by weathering of primary silicates in soil may be leached out or is consumed by the formation of secondary silicates. It may precipitate as amorphous silica, be adsorbed by pedogenic oxides and hydroxides, or be taken up by plants (Sommer et al., 2006). The plants exclusively take up Si as monomeric silicic acid (H4SiO4; pKa1 = 9.8; Haynes, 2014), which is transported into plant organs, where it polymerizes as hydrous amorphous silica or bioopal, so-called phytoliths. During the decomposition of plants, bioopal Si is returned into soil (Kaufman et al., 1981) so that a large proportion of amorphous silica in most soils is comprised by bioopal (Drees et al.,
⁎
1989). Earthworms play an important role in the improvement of soil physical conditions and plant growth (Winding et al., 1997; Scheu, 2003). They regulate the soil structure by creating burrows, which facilitate water and gas transport, by mixing soil minerals with organic material (bioturbation) and by creating aggregates (Winding et al., 1997). Earthworms accelerate the degradation of soil organic matter (SOM) by increasing its available surface area by comminution and fostering soil microbial activity (Blouin et al., 2013). Earthworms induce both chemical and physical weathering of minerals, for instance by promoting the transformation of clay minerals and mechanical stress (Suzuki et al., 2003; Carpenter et al., 2007). Small mineral particles in the earthworm gut contribute to grinding of plant material as well as physical weathering of minerals in the muscular gizzard (Schulmann and Tiunov, 1999; Marhan and Scheu, 2005). Bacteria living in the earthworm gut enhance the solubility of silicates (Hu et al., 2018). The authors carried out experiments with earthworm-gut bacteria and demonstrated that inoculation of potting soil with these bacteria promoted growth of maize plants. Bityutskii et al. (2016) reported that earthworms increase the content of water-extractable (thus plant-
Corresponding author. E-mail address: [email protected] (A. Georgiadis).
https://doi.org/10.1016/j.pedobi.2019.05.001 Received 20 December 2018; Received in revised form 2 May 2019; Accepted 7 May 2019 0031-4056/ © 2019 Elsevier GmbH. All rights reserved.
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available) Si in soil after passage through the earthworm gut. However, up to now the source of the additional plant-available Si provided by actions of earthworms is unknown. Other Si fractions such as adsorbed Si, Si bound to SOM, Si occluded in pedogenic oxides and amorphous silica are potential sources of plant-available Si after mobilization from these fractions and its redistribution induced in the earthworm gut. Thus, these fractions may act as important sources for dissolved Si that can be taken up by plants. Therefore, the knowledge about the potential of earthworms to increase Si availability can be important for sustainable soil management. The aim of this study was to clarify the effects of earthworms on the release of Si from soil constituents and on the distribution of Si among operationally defined fractions in soil. We hypothesize that chemical, biological and physical weathering of Si-containing minerals together with transformation of SOM during the passage through the earthworm gut lead to increasing Si solubilization in soil and to a redistribution of Si among fractions. We expect an increase in the amounts of Si in more mobile fractions at the expense of Si in less mobile fractions after the passage of soil through the earthworm gut. For a quantification of Si among fractions, we applied a sequential Si extraction procedure (Georgiadis et al., 2013) on selected soil samples to quantify Si in soil in mobile and adsorbed (plant-available) form, Si bound to SOM, Si occluded in pedogenic oxides and amorphous silica before and after the passage of soil through the earthworm gut. In additional independent experiments, we checked the effects of earthworms on the release of Si from models of Si-containing soil constituents. These model substances comprised quartz, bioopal and wheat straw. We used quartz as a model of a crystalline Si form, bioopal as a model of amorphous Si and wheat straw as model of Si-containing plant material in soil. Using defined model substances may help to explain the findings from the experiments with soil and thus to differentiate the superimposing processes that occur during the (re-) distribution of Si in a complex mixture like soil. We tested the effects of endogeic earthworms on the distribution of Si among fractions on soil samples from three sites with different land use, representing different earthworm habitats. We used endogeic earthworms, as they are abundant in the studied areas. As geophagous species, they contribute to silicate weathering and Si mobilization in soil (Bityutskii et al., 2016; Hu et al., 2018). In the approach with model substances, we used epigeic earthworms, as they strongly affect transformation of organic matter and may thus mobilize Si (Sims and Gerard, 1999; Bityutskii et al., 2016).
Table 1 Selected properties of soil samples used for the experiments with Octolasion cyaneum Savigny (experiment a). Soils
Horizon
Corga Nb [g kg−1]
Sand
Silt %
Clay
pHc
pHc (casts)
Stagnic Luvisol Haplic Luvisol Haplic Umbrisol
Ap Ah Ap
9.0 46.7 36.2
3 2 7
72 76 64
25 22 29
6.0 3.9 5.0
6.2 3.9 5.0
a b c
1.1 3.6 3.1
Organic carbon. Total nitrogen. Measured in CaCl2 solution at a solid-to-solution ratio of 1:10.
The model substances (Table 2) were mixed with peat that was used as an experimental matrix for feeding earthworms to ensure their survival in the experiments. Pure peat served as control. 2.2. Experimental setup We carried out two sets of experiments - (a) with soil samples and the endogeic earthworm species Octolasion cyaneum Savigny that is geophagous and lives mostly in the upper mineral soil, and (b) with model substances and the epigeic species Eisenia andrei Bouché that thrives in compost and manure and feeds almost exclusively on organic matter (Sims and Gerard, 1999). O. cyaneum were collected by the Thielemann octet method and hand sorting from a garden near Stuttgart (Germany). E. andrei species were collected by hand sorting from compost. Prior to the experiments, O. cyaneum species were kept 72 h in fresh soil for adaptation. E. andrei were kept for one week in peat for adaptation and then depurated in moistened Petri dishes for 24 h for defecation, before being added to the mixtures of model substances with peat. For experiment (a), we placed about 15 g of each soil sample in a Petri dish (in total, six Petri dishes per sample) and adjusted the gravimetric water content to 36–38%. Subsequently, one O. cyaneum specimen was placed into each of three Petri dishes per sample. A further three Petri dishes were left without earthworms as a control (i.e., three replicates with earthworms and three control replicates for each sample; in total 18 Petri dishes). The earthworm casts after single ingestion, produced after two days were collected, dried at 30 °C and stored in plastic vessels. For experiment (b), we mixed 12 g of wheat straw (milled to powder), 7 g of quartz and 5 g of bioopal separately with peat (5–12 g, depending on the model substance; Table 3). We placed each mixture in a Petri dish (in total four Petri dishes per sample). A further four Petri dishes contained only peat (control). Subsequently, we placed E. andrei in two Petri dishes per sample (20 specimens per Petri dish). A further two Petri dishes were left without earthworms (two replicates with and two replicates without earthworms for each mixture and pure peat; in total 16 Petri dishes). The water content of the mixtures was adjusted based on the earthworms’ activity in the Petri dishes to achieve optimum conditions for their adaptation (Table 3). After 24 h, we rinsed the earthworms with water and placed them in a separate Petri dish until the earthworms produced casts. Then, we dried the casts at 30 °C and stored them in plastic vessels. We did not use glassware to minimize Si contamination.
2. Materials and methods 2.1. Soils and model substances We sampled the A horizons from soil profiles on three study sites that represent typical sites of arable land, grassland and forest of southwest Germany. Three replicates per site (each 500 g) were pooled to one composite sample per site. The soils comprised a Stagnic Luvisol (SL; WRB classification (IUSS Working Group WRB, 2015)) developed from loess (arable land), a Haplic Umbrisol (HU) developed from colluvial loam (grassland, formerly arable land) and a forested Haplic Luvisol (HL) developed from loess. The soil samples were passed through a 2-mm sieve and stored at 5 °C. Particle-size distribution was determined by wet sieving (sand) and sedimentation (silt and clay). For consistency, we determined the pH of both, the soil samples and the earthworm casts, with a glass electrode in 0.01 M CaCl2 at a solid-to-solution ratio (SSR) of 1:10, as the amount of earthworm casts obtained in experiments was scarce. Total carbon (C) and nitrogen (N) contents of the soils were determined by elemental analysis (dry combustion) (Vario MACRO, Elementar, Hanau, Germany). The soil samples were free of carbonates, thus the contents of soil organic C equaled those of total C. Soil properties are summarized in Table 1.
2.3. Analyses After drying (35 °C) the soil samples, model substances with peat and pure peat as well as the respective earthworm casts, we applied a sequential extraction scheme (Georgiadis et al., 2013) to quantify Si in different fractions, which included: 1) Mobile Si (Sil), extracted by 0.01 M CaCl2, SSR (solid-to-solution ratio) 1:10, 2
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Table 2 Model substances used for the experiments with Eisenia andrei Bouché (experiment b). Substance
Characteristics
pHa
Corg
Peat Quartz Bioopal Wheat straw
< 2 mm, culture substrate TKS® 1 H 32, average particle size 320 μm from Equisetum arvense harvested from arable land near Stuttgart Triticum aestivum, harvested from arable land
6.2 5.6 4.0 6.1
480 n.d. 16.2 427
N [g kg−1] 9.98 n.d. 0.29 4.86
Sitotalb
Source
0.14 n.d. n.d. 6.5
Floragard, Oldenburg, Germany Quarzwerke Frechen, Germany obtained after Bartoli and Wilding (1980) –
n.d. not determined. a Measured in H2O at a solid-to-solution ratio of 1:25. b Quantified after Georgiadis et al. (2015).
2) Adsorbed Si (Siad), extracted by 0.01 M acetic acid, SSR 1:10, 3) Si bound to SOM (Siorg), released by concentrated H2O2 at 85 °C, SSR 1:30, 4) Si occluded in pedogenic oxides and hydroxides (Siocc), released by 0.2 M NH4-oxalate under UV radiation, SSR 1:50 and 5) Si in amorphous silica (Siam), extracted by 0.2 M NaOH after 168 h, SSR 1:400.
2.4. Statistics
For experiment (b), we restricted the extraction to steps 1–3 and 5 (SSR 1:20 in steps 1 and 2, and 1:60 in step 3), as we did not use any oxide as model substance. Samples with bioopal were not subjected to step 5, since bioopal is completely dissolved in this step, even without passage through the earthworm gut (Georgiadis et al., 2015). The SSRs were adapted for optimal Si extraction. All extracts were centrifuged (1400 g) until the supernatant was clear and then filtered through paper filters (1–2 μm). Silicon in the extracts was analyzed by microwave plasma-atomic emission spectrometry (4200 MP-AES, Agilent, Waldbronn, Germany). MP-AES, like ICP-AES and -OES (inductively coupled plasma optical emission spectrometry), bases upon the release of element-specific electromagnetic radiation from excited atoms. In an MP-AES system, N2 fuels a microwave plasma utilized for excitation. We analyzed Si using its most sensitive atomic line with negligible spectral interferences at 288.158 nm. Atomic lines are less susceptible to matrix effects (Karlsson et al., 2015). Quality control included calibration and measuring samples with known concentration for each series of measurements in the respective matrix (extractant solution). The precision in all Si analyses was < 5% (relative standard deviation). The detection limit for Si in 0.01 M CaCl2 (the extractant with the lowest Si concentrations measured) was 20 μg l−1. The amounts of extracted Si were related to the material dried at 30 °C. The amounts of Si extracted from the model substances (experiment (b)) were corrected for the Si amounts determined in pure peat obtained in identical experiments. About 5–10 particles from each experiment (b) were mounted on an Al sample holder with double-sided adhesive and conductive carbon tape, sputtered with a thin layer of Au/Pd alloy. We analyzed them by scanning-electron microscopy (LEO 420, LEO Electron Microscopy, Cambridge, UK) in secondary-electron mode to visualize particles before and after their passage through the earthworm gut.
3. Results
We used SigmaPlot 11.0 (Systat Software Inc., San Jose, USA) for statistical analyses. We used the Spearman rank correlation coefficient (rs) to correlate soil data with non-linear distribution. Levels of statistical significance were *** P ≤ 0.001, ** 0.001 < P ≤ 0.01, * 0.01 < P ≤ 0.05 and not significant when P > 0.05.
3.1. Experiment (a) - Effects of endogeic earthworms on the fractionation of Si in soil Relative to the earthworm-free soil samples, the sum of Si extracted from the earthworms’ casts in the five extraction steps increased by 10% and 28% (SL and HL, respectively), but decreased negligibly by 2.5% (HU, Fig. 1a). The passage through the earthworm gut hardly affected the contents of mobile Sil and adsorbed Siad in all soil samples. The amounts of Sil and Siad extracted from the casts increased by 4% and 2% (SL), and decreased by 4.5% and 5% (HU) and by 5% and 4% (HL), respectively, relative to the earthworm-free samples (Fig. S1). Overall, Sil and Siad contents were smallest among all Si fractions. The Sil contents were considerably lower for the SL samples (7.4 and 7.7 mg kg−1 for the earthworm-free samples and casts, respectively; Table S1) than for the HU (11.8 and 11.2 mg kg−1 for the earthworm-free samples and casts, respectively) and HL samples (12.5 and 11.9 mg kg−1 for the earthworm-free samples and casts, respectively). The Siad contents were significantly larger in SL samples (98 and 100 mg kg−1 for the earthworm-free samples and casts, respectively) than in the HU (29 and 27 mg kg−1 for the earthworm-free samples and casts, respectively) and HL samples (30 and 29 mg kg−1 for the earthworm-free samples and casts, respectively). The Sil contents were positively correlated with c (H+) (rs = 0.8***) and the contents of Corg (rs = 0.8***) and N (rs = 0.8***). The Siad contents of the earthworm-free soil were negatively correlated with c(H+) (rs = -0.9***) and the Sil (rs = -0.8***), Corg (rs = -0.9***) and N (rs = -0.9***) contents. The Siad contents of the casts were not correlated with any other soil parameter. The contents of Si bound to SOM were positively affected by O. cyaneum. Extraction of Siorg from the casts increased by 7%, 19% and 41% (SL, HU and HL, respectively, Fig. 1b). The Siorg contents of all samples were strongly negatively correlated with the clay contents (rs = -0.9***).
Table 3 Properties of model substances and experimental conditions used in experiments with Eisenia andrei Bouché. Substance
Mass ratio of model substance to peat
Quartz mixed with peat Bioopal mixed with peat Wheat straw mixed with peat Pure peat
1:1 1:1 1:2 –
a
Mass per Petri dish [g] 14 10 18 10
Measured in H2O at a solid-to-solution ratio of 1:25. 3
Water content [wt%] 125 300 300 300
pHa
pHa (casts)
6.5 5.9 6.4 6.3
7.2 7.3 7.7 7.5
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Fig. 1. Experiment (a): Contents of Si extracted from soil before (white) and after (grey) the passage through the gut of Octolasion cyaneum Savigny according to sequential extraction (SL, Stagnic Luvisol; HU, Haplic Umbrisol; HL, Haplic Luvisol): a) total Si extracted; b) Si bound to soil organic matter; c) Si from amorphous silica and d) Si occluded in pedogenic oxides. Data are expressed as arithmetical means ± standard deviation (n = 3).
extraction from bioopal were distinctly smaller, with a decrease by 16% (first step), and increases by 8% (second step) and 95% (third step), respectively (Fig. 2c). The smallest total contents of Si extracted were found for the quartz samples (0-0.14 mg g−1; Table S2) and the largest (1.2–5.4 mg g−1) for the bioopal samples. Treatment with H2O2 released the largest amounts of Si from all samples. Extraction with 0.2 M NaOH (fifth step) released no Si from quartz in both experiments, and relatively small amounts from wheat straw (0.91 and 0.93 mg g−1 for the undigested samples and casts, respectively). The largest changes in the distribution of Si among forms were found for quartz. The casts exhibited an increase of Si shares by six and 34 percentage points after extraction in the first and second step, respectively, and a decrease by 40 percentage points after extraction with H2O2 in the third step. The Si shares of the casts of the wheat-straw samples increased by three percentage points in the first and second extraction steps and decreased by three percentage points in the third and fifth extraction steps, respectively. The redistribution of Si shares in the samples with bioopal was negligible. Again, it should be considered that the results of this experiment are also based on single ingestion of model substances by earthworms. The passage of all model substance mixed with peat through the gut of E. andrei increased the pH by 0.7 (quartz), 1.4 (bioopal), 1.3 (wheat straw) and 1.2 (pure peat).
The contents of Siocc extracted from the casts were larger than extracted from the earthworm-free samples with increases of 8%, 6% and 38% (SL, HU and HL, respectively, Fig. 1d). In contrast to Siorg, the Siocc contents of all samples were positively correlated with clay contents (rs = 0.7*). The contents of Si from amorphous silica were larger for the casts (increase of 13% and 29%) of the SL and HL samples and lower for the casts of the HU samples (decrease of 5%), relative to the earthwormfree samples. Silicon in amorphous silica constituted the largest Si fraction, making up 1.8–2.6 mg g−1 (Fig. 1c). The Siam contents of all samples were positively correlated with clay contents (rs = 0.9***) and negatively with the Siorg contents (rs = -0.8***/** earthworm-free/ cast). The largest changes in the distribution of Si among the fractions were found for the HL samples after their passage through the earthworms’ guts. Relative to the earthworm-free treatment, the share of Siorg in the total extractable Si increased by 2.1 percentage points, while that of Siam and Siad decreased by 1.5 and 0.7 percentage points, respectively. In the casts of the HU samples, the shares of Siorg and Siocc increased by 1.3 and 1.1 percentage points, respectively. Changes in the distribution of Si among fractions after passage through the earthworm gut were negligible for the SL samples. The pH of the SL samples (6.0–6.2) was slightly increased in casts of O. cyaneum, while pH values of other samples were not changed after the passage through the earthworm gut relative to the earthworm-free soils (Table 1). However, we have to keep in mind that all these results are based on single ingestion of soil by earthworms and might become more pronounced when repeated, on the time scale of years.
4. Discussion 4.1. Effects of earthworms on contents of plant-available Si and Si bound to SOM
3.2. Experiment (b) - Effects of epigeic earthworms on Si extraction from model substances in a peat matrix
We detected an increase in the contents of Si extracted from soils and model substances after the passage through the earthworm gut. This increase was especially pronounced for the HL samples, wheat straw and quartz. Silicon bound to SOM was the major contributor to the additional extractable Si from soils. The largest increase in the contents of Siorg (by 41%) was detected for the HL samples with the largest organic C content among the soils under study. An increase in the contents of Siorg in the casts of all soil samples and an increase in total extractable Si in the casts of the wheat straw indicate that earthworms transformed SOM (Drake and Horn, 2007). Transformation and degradation of SOM during the passage of the earthworm gut induce the release of Si that is bound to SOM and make it more easily
Unlike for the bioopal samples, the total amounts of Si extracted from the model substances increased after passage through the earthworm gut, by 257% for quartz and by 20% for wheat straw, relative to undigested samples (Fig. 2a). Specifically, Si extracted from the casts of the quartz samples increased by 764% in the first step (extracted by CaCl2), 1730% in the second step (acetic acid) and 95% in the third step (H2O2), relative to the undigested samples (Fig. 2b). E. andrei had smaller effects on Si extraction from wheat-straw samples with increases in Si extraction by 71%, 38%, 13% and 3% in the first, second, third and fifth step, respectively. The earthworm-induced effects on Si 4
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Fig. 2. Experiment (b): Contents of Si extracted from model substances mixed with peat before (white) and after (grey) the passage through the gut of Eisenia andrei Bouché according to sequential extraction: a) total Si extracted; b) quartz; c) bioopal and d) wheat straw. Step 1: extracted by 0.01 M CaCl2; Step 2: extracted by 0.01 M acetic acid; Step 3: released by concentrated H2O2; Step 5: extracted with 0.2 M NaOH. Pure peat was used as control. Data are expressed as arithmetical means ± standard deviation (n = 2). Please note the different scales on the y-axes.
aggregates (Emerson et al., 1986). We detected such aggregates in the earthworms’ casts of quartz (Fig. 3a) mixed with peat as patches of peat particles adhered to quartz grains (Fig. 3b). Additionally, during the passage through the earthworm gut, minerals may be mechanically altered. Suzuki et al. (2003) detected finer and rounded feldspar and quartz grains after the passage of earthworms. Extractability of Si from such mineral grains and from the scraped material should increase because of an increase in the surface area, relative to undigested mineral grains. Thus, mechanical weathering of quartz particles, together with an increase in solubilization induced by microbial activity, may explain the large increase in the amounts of Si extracted from the casts of the quartz samples in our study. Therefore, earthworms contribute to mobilization of plantavailable Si from quartz and very likely other Si-containing minerals. However, the contribution of plant-available Si from quartz in soil is presumably small. Sommer et al. (2002) roughly estimated the production of cast by earthworms to 5 kg m−2 a-1 for soil in the area under study. Assuming that quartz dominates the sand fraction (accounting for 2–7% of the total soil; Table 1), 15–50 mg plant-available Si m−2 a-1 is provided. This amount of Si, provided by quartz, corresponds to 5–8% of plant-available Si (as the sum of Sil and Siad) that is present in the earthworms’ casts produced in the experiments with soil. Apart from the general low susceptibility of quartz to physical and chemical weathering, the small contribution of quartz to plant-available Si resulted from single ingestion of soil by earthworms, which could increase after repeated ingestion on a larger time scale.
extractable (Bityutskii et al., 2016). Furthermore, Si released from the earthworm casts of the soil samples by H2O2 may partially represent mobile and adsorbed Si protected within aggregates and thus not accessible for extraction in the first two extraction steps. This was indicated by decreased contents of Sil and Siad in the casts of HU and HL. Earthworms produce compact aggregates during digestion (Jouquet et al., 2008). We detected such aggregates in the casts of model substances mixed with peat (Fig. 3). The aggregates may have been formed by glue produced by gut bacteria or by reinforcement by fibrous plant residues (Lee and Foster, 1991). Oxidizing the aggregating SOM by H2O2 may have led to the dispersion of aggregates so that Sil and Siad previously present in the aggregates may have been released into solution. The strong negative correlation between the contents of Sil and Siad and the strong positive correlation of the contents of Sil with the Corg and N contents in our study point to an association of these Si species with SOM. The partly negative to negligible effect of O. cyaneum on the extraction of mobile and adsorbed Si is in contrast to the findings by Bityutskii et al. (2016). They detected an increase in the contents of water-extractable Si after the passage of soil through the guts of a endogeic earthworm species, Aporrectodea caliginosa. Like in our approach, Bityutskii et al. (2016) determined the Si contents in the earthworm casts after single ingestion of soil. Moreover, the Si firstly released from Si-containing minerals (such as silicates or amorphous silica) and then adsorbed on SOM in the earthworms’ guts, may have contributed to the enhanced contents of Siorg in the earthworms’ casts. Silicon transferred to SOM may then be released by oxidation of SOM by H2O2 treatment. The increase in the Siorg shares in the casts of HL and HU samples was mostly at the expense of Si from amorphous silica. This is also indicated by a close negative correlation between the contents of Siam and Siorg.
4.3. Effects of earthworms on the contents of Si in amorphous silica Earthworms hardly affected the solubilization of Si from bioopal. The solubility of amorphous silica increases with increasing pH (Fraysse et al., 2009). Usually, the pH of earthworm gut is neutral to slightly alkaline (6.4–7.7) so that homeostasis occurs (Drake and Horn, 2007). E. andrei increased the pH of the casts of all model substances (Table 3). However, slightly less Si was extracted from the casts of bioopal mixed with peat in the first extraction step. This may be explained by a protective effect of the peat: After passage of the bioopal-peat mixture through the earthworm gut, coarse bioopal-peat aggregates have formed (Fig. 3c, d). The small bioopal particles are embedded into coarser peat structures and are thus less available for extraction. These aggregates were destroyed during the first extraction step by shaking the samples, resulting in slightly larger Si release in the second
4.2. Effects of earthworms on the release of Si from soil minerals Microorganisms living in the earthworm gut may promote weathering of Si-containing minerals and increase Si extractability (Hu et al., 2018). Microbes can colonize mineral surfaces. Protons, extracellular enzymes, metabolic by-products, organic acids and chelators, produced by microbes may induce dissolution of minerals in the earthworm gut (Bennett et al., 2001; Uroz et al., 2009; Hu et al., 2018). These microbial products can interact with the surfaces of mineral particles and contribute to the formation of organo-mineral complexes and 5
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Fig. 3. Secondary electron images of quartz (a, b), bioopal (c, d) and wheat straw (e, f) mixed with peat before (a, c, e) and after (b, d, f) the passage through the gut of Eisenia andrei Bouché. The white scale bar represents 20 μm. Q, quartz; B, bioopal; W, wheat straw; P peat.
4.4. Effects of earthworms on the contents of Si occluded in pedogenic oxides
extraction step. This is supported by the changes in Si shares after passage of bioopal through the earthworm gut, relative to the undigested bioopal: The share of Si in the cast decreased after the first extraction step and increased after the second and third. Nonetheless, the effect of earthworms on Si extraction from bioopal was the smallest among all model substances and may be considered negligible for the mobilization of Si. In the experiments with soils, the pH values of the casts did not change. However, the amounts of Siam extracted from the casts of SL and HL samples were distinctly larger than extracted from the earthworm-free samples. We detected the largest effect of earthworms on Siam extraction from the HL samples of the forested site (increase of Siam in the casts by 29%). Usually, bioopal is the most abundant form of amorphous silica in topsoil (Georgiadis et al., 2014), mostly as phytolith. In contrast to the agricultural sites, bioopal particles in the forested soil were not crushed mechanically by soil cultivation, e.g. by ploughing. Thus, these relatively large bioopal particles can be crushed into smaller particles by muscular contractions of the earthworms’ gizzard (Edwards and Bohlen, 1996). Subsequently, these particles became more easily extractable by NaOH. Thus, it is likely that the mechanical stress during the passage of the earthworm gut is more important for bioopal-Si mobilization than the change in pH in the earthworm gut. However, the effect of the gut milieu may become more pronounced after repeated passage of bioopal through the earthworm gut.
Bacteria of the earthworm gut may participate in redox reactions. Pseudomonas and Bacillus species have been detected in the gut of endogeic earthworms (Drake and Horn, 2007; Hu et al., 2018). They can reduce Mn(IV) and Fe(III) (Sundaram et al., 2012) and thus may induce partial reductive dissolution of Fe/Mn oxides, which are important sorbents of Si species (Georgiadis et al., 2013, 2017), and of Fe(Mn)containing silicates, which would induce the release of bound Si into the earthworm gut. After excretion of the casts and exposure to the atmosphere, Fe and Mn oxides may subsequently precipitate and retain previously released Si by adsorption, (co-)precipitation or occlusion. Silicon from other sources released in the earthworm gut (e.g., Si from amorphous silica) may be involved in these sorption processes as well. This may help to explain the higher amounts of Siocc extracted from the earthworm casts (increase up to 38%) as well as the changes in the distribution of Si shares in the casts of HU and HL samples with increase in Siocc and decrease in Siam. 5. Conclusions Our study demonstrates that earthworms (O. cyaneum and E. andrei) affect the distribution of Si among fractions by the passage of soil through their gut, even after a single ingestion. They increased the 6
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solubilization of Si in soil, inducing the redistribution of Si among the fractions. Earthworms induced the formation of aggregates, possibly occluding plant-available Si. On the other hand, they induced a slight mobilization of plant-available Si from quartz. However, the proportion of Si extractable from quartz in soil casts was low, amounting to 5–8% of the whole plant-available Si in soil casts. The major contributor to the additional extractable Si from the casts was Si bound to SOM, indicating transformation of SOM by earthworms. The Si occluded in oxides and hydroxides as well as amorphous silica also contributed to the increase of extractable Si from casts. The sum of extractable Si from all analyzed fractions in the casts may be considered as potentially plant-available Si. Thus, earthworm casts may act as an important source for this kind of Si in soil, with larger contents than the surrounding soil. The Si may become available for plants after destruction of the aggregates. However, stability of such aggregates as well as the Si release in the soil solution after their destruction has not yet been studied. The role of such aggregates for the long-term Si release and retention is neither clarified. On the one hand, Si release from mineral particles after their passage through the earthworm gut increases due to mechanical stress or microbially induced weathering. On the other hand, organic matter, covering or embedding these particles can oppositely pose as sorbent for previously released Si. These open questions may be answered in long-term experiments that offer the possibility of repeated ingestion of soil by earthworms.
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