Effects of soil macrofauna on other soil biota and soil formation in reclaimed and unreclaimed post mining sites: Results of a field microcosm experiment

Applied Soil Ecology 33 (2006) 308–320 www.elsevier.com/locate/apsoil

Effects of soil macrofauna on other soil biota and soil formation in reclaimed and unreclaimed post mining sites: Results of a field microcosm experiment Jan Frouz a,*, Dana Elhottova´ a, Va´clav Kura´zˇ b, Monika Sˇourkova´ a,c a

Institute of Soil Biology, Biological Centre Academy of Sciences of the Czech Republic, Na Sa´dka´ch 7, Cˇeske´ Budeˇjovice, CZ 37005, Czech Republic b Faculty of Civil Engineering, Czech Technical University, Thakurova 7, Praha 6, CZ 16629, Czech Republic c ˇ eske´ Budeˇjovice, CZ 37005, Czech Republic Faculty of Biological Sciences, University of South Bohemia, Branisˇovska´ 31, C Received 11 February 2005; received in revised form 1 November 2005; accepted 3 November 2005

Abstract The effect of macrofauna on soil organic matter accumulation (carbon and nitrogen content in the soil), soil hydraulic properties (water holding capacity, water field capacity and wilting point), microbial respiration, microbial biomass, composition of microbial community (using PLFA) and density of soil mesofauna was studied in a field microcosm experiment. Microcosms were located in two sites, either in a reclaimed or unreclaimed, naturally revegetated post mining site, in the Sokolov brown-coal mining area (Czech Republic) for 1 and 3 years. Both sites were located on tertiary clay material; the reclaimed site was covered by a 20–30year-old alder (Alnus glutinosa and A. incana) plantation; the non-reclaimed site was about 20 years old and covered by shrubs dominated by Salix caprea. The field microcosms consisting of litter (autochthonous litter) and mineral (tertiary clay from a pioneer site) layer were exposed on these sites for 1 and 3 years. The microcosms were either accessible or non-accessible to soil macrofauna. The access of soil macrofauna did not increase significantly carbon mineralization (the loss of organic matter from the whole microcosms) but increased the translocation of organic matter into the mineral layer. This effect seemed to be more pronounced in the reclaimed site. Accumulation of organic matter in the mineral layer resulted in higher microbial respiration and biomass and in increased water retention in the soil. It is considered that these effects correspond with litter fragmentation and soil mixing by soil saprophagous macrofauna. # 2005 Elsevier B.V. All rights reserved. Keywords: Fauna; Soil formation; Microbial community; PLFA; Decomposition; Carbon storage

1. Introduction Open cast coal mining causes massive disturbance of ecosystems at a landscape level. For example, the total area which will be disturbed at the end of mining activities (around 2036) in the Sokolov coal mining

* Corresponding author. Tel.: +420 38 7775769; fax: +420 38 5300133. E-mail address: [email protected] (J. Frouz). 0929-1393/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2005.11.001

district (one of two major mining districts in the Czech Republic) will reach more than 6000 ha. The majority of this area (75%) is dedicated for forest reclamation. Disturbance caused by open coast mining is not only large scale but at the same time very intensive. Spoil material overlying the coal layer is removed and deposited in heaps. The largest heaps are thousands of hectares in area and reach elevations of more than 100 m above the original terrain; hence the original ecosystems affected by this activity are completely destroyed (excavated or buried). The material excavated

J. Frouz et al. / Applied Soil Ecology 33 (2006) 308–320

from up to 200 m depth differs substantially from normal soils. Typically, spoil material does not contain recent organic carbon, but may contain some fossil carbon (Krˇ´ıbek et al., 1998). Adverse properties of such material including sensitivity to erosion, unsuitable water regime, nutrient deficiency, etc., may reduce plant growth in some post mining landscapes (Bradshaw, 1993; Scullion and Malinovszky, 1995). Thus, reconstruction of soil functions is necessary for ecosystem restoration in post mining sites (Bradshaw, 1997; Frouz et al., 2001a). In some post mining sites soil reestablishment is accelerated by spreading of topsoil (Stewart and Scullion, 1989) on the tailings. In other post mining areas, including the sites where this study was conducted, no topsoil is added and soil develops denovo from raw spoil material. Soil formation in post mining sites is a complex process, including mouldering of geological substrate, physical, chemical and biological changes. Here, we focus on one particular process, accumulation of soil organic matter in mineral soil. Soil organic matter has many important functions in the soil; it is a source of energy for soil microflora, affects sorption capacity and water holding capacity of soil, supports formation of soil aggregates and soil structure, etc. (Allison, 1973; Insam and Domsch, 1988; Malik and Scullion, 1998; Stewart and Scullion, 1988, 1989). This is particularly important in situation when no topsoil is used to cover raw spoil material dumped in colliery soil heaps which typically have little recent organic matter (Sˇourkova´ et al., 2005). During succession the amount of organic matter stored in the soil increases (Schafer et al., 1979; Sˇourkova´ et al., 2005). Vertical distribution of organic matter in the soil changes as well: in the initial stages of succession most of the organic matter is located in litter on the soil surface, later on during succession more organic matter is incorporated into mineral soil (Schafer et al., 1979) and an organo-mineral humus layer is formed. The incorporation of organic matter into mineral soil also supports many soil organic matter functions such as water holding capacity and nutrient sorption (Allison, 1973). The accumulation of soil organic matter depends on the amount and quality of organic matter entering the soil and on the soil biota, which can affect the rate of decomposition and the spatial distribution of organic matter in the soil (Lavelle et al., 1997; Scullion and Malik, 2000; Marashi and Scullion, 2003; Smith and Bradford, 2003). Individual groups of soil biota may play different roles in this process. Soil microflora are responsible for the bulk of the decomposition activity in the soil. On the other hand, soil macrofauna affect fragmentation, aggregation and mixing of organic

309

matter and are responsible for the physical modification and spatial distribution of soil organic matter (Lavelle et al., 1997). In this way, these animals also indirectly affect the activity of soil microflora (Anderson and Ineson, 1984; Lavelle et al., 1997; Scullion and Malik, 2000; Marashi and Scullion, 2003). There are several methods for measuring effects of various groups of soil biota on litter decomposition, among which litterbags with various mesh sizes are among the most popular (Irmer, 1995; Scheu and Wolters, 1991). However, as pointed out by Wachendorf et al. (1997), mass loss from litterbags with various mesh sizes may be interpreted in different ways. In litterbags not accessible to soil fauna, the majority of mass loss can be explained by microbial respiration and hence represents carbon (C) mineralization. By contrast, in litterbags with large mesh sizes, only a small fraction of litter loss represents mineralization, while the majority of this loss can be accounted for by translocation of organic matter from the litter to the mineral layer. To measure both processes at once, we adopted a microcosm technique, which included litter and mineral layer (Frouz, 2002). This allows us to measure the effect of fauna on both mineralization and mixing of organic matter with mineral soil. The role of macrofauna in soil mixing may be particularly important in post mining sites (Scullion and Malik, 2000; Marashi and Scullion, 2003), especially in sites covered by forest because soil macrofauna may be very abundant there (Topp et al., 1992; Frouz et al., 2001a) and because, in forest, tillage or other techniques which may accelerate soil mixing cannot be applied. The effect of macrofauna may be affected by many other factors among which the vegetation cover is the most important. Vegetation not only supplies the bulk of organic material entering in the soil, but also indirectly determines the composition of the soil fauna (Lavelle et al., 1997). Typically, vegetation producing a large quantity of easily decomposable litter supports higher abundance of macrofauna, whereas vegetation producing a low amount of hardly decomposable litter supports few soil macrofauna but a high density of soil mesofauna (Lavelle et al., 1997). This pattern was also well documented in post mining sites (Frouz et al., 2001a,b, 2002). Earlier studies in the Sokolov coal mining area found that the development of the soil humus layer is slower in naturally revegetated sites, in comparison with the reclaimed sites (Frouz et al., 2004). The unreclaimed sites had poorly developed macrofaunal communities. On the other hand, the reclaimed sites were occupied by abundant macrofauna and a well developed earthworm community (Frouz et al., 2001a,b). In this study, we tested the hypothesis that differences in the soil macrofaunal composition may

310

J. Frouz et al. / Applied Soil Ecology 33 (2006) 308–320

contribute to different rates of soil forming process in these sites. In particular, we explored the question of how soil macrofauna can affect soil mixing (removal of organic matter from the litter layer and its incorporation into the organo-mineral humus layer) and organic mater mineralization in two post mining sites with different litter quality. In addition, we studied the effect of macrofauna mediated soil mixing on other selected soil parameters. 2. Material and methods 2.1. Study sites The study was conducted during 1999–2002 in the Sokolov coal mining area in North Bohemia (508140 2100 N, 128390 2400 E). The average altitude of the study area is 600 m a.s.l., the mean annual precipitation is 650 mm and the mean annual temperature 6.8 8C. See Frouz et al. (2001a) for more details on the study area. During open coast mining near Sokolov, a large amount of spoil is excavated from the depth up to 200 m and stored in large heaps outside the mining pit. The majority of this spoil material in the Sokolov area is formed by alkaline (pH 8) tertiary clay material of the so-called cypris formation. Two sites were selected for this study; both were located on cypris clay spoil, on one large spoil heap, about 1 km apart and both were covered with ca. 20-year-old vegetation. The first was reclaimed by planting alder (a mixture of Alnus glutinosa and A. incana) 20 years ago, when the spoil heap was 5–10 years old, i.e. the site is 25–30 years old. The second site was heaped about 20 years ago and no reclamation effort was applied. It was covered by vegetation, which developed by natural colonisation and is dominated by willow shrubs (Salix caprea). No levelling was applied on unreclaimed sites and longitudinal rows of depressions and elevations formed by the heaping process remained here. Further characteristics of the unreclaimed site refer to the depression where microcosms were exposed. A ca. 2 cm deep fermentation and ca. 4 cm deep humus layers were developed on the reclaimed site. In the unreclaimed site, a 4–6 cm deep fermentation layer was developed, while the humus layer was missing. The density of soil saprophagous macrofauna was much higher in the reclaimed than in the unreclaimed site (Frouz et al., 2004). Based of sampling conducted on the sites where the microcosms were exposed the density of the Lumbricidae was 152
35 individuals m2 in the reclaimed site and 11
16 individuals m2 in the unreclaimed site. The density of dipteran larvae was

127
75 and 44
27 individuals m2 and the density of millipedes was 15
6 and 4
4 individuals m2 in reclaimed and unreclaimed sites, respectively (Frouz et al., 2004) 2.2. Microcosm preparation and exposure Two types of microcosms were prepared, accessible for macrofauna and non-accessible for macrofauna. The microcosms were similar to those used by Frouz (2002). They consisted of plastic boxes 18 cm  25 cm  5 cm with 12 openings (1 cm in diameter) covered with 0.2 mm nylon net on the top and bottom surfaces. In macrofauna-accessible treatments, six horizontal openings (4 mm  30 mm) were located in each longer lateral side; no such openings were made in the nonaccessible treatments. Each box contained a mineral layer (100 g of clay spoil) and a litter layer (autochthonous litter, ca. 60 g of dry weight equivalent). A 2 mm nylon mesh with eight large (1 cm diameter) openings separated the layers. This net was pervious to all size groups of soil macrofauna and served only to mark the original border between the litter and the mineral layer. The spoil used in the mineral layer came from a pioneer site (ca. 10-year-old), where vegetation was almost absent, from a depth of 3–10 cm. The clay was sieved through a 5 mm screen and homogenized. The litter was hand sorted, cut into pieces ca. 1 cm  3 cm and homogenized. Both litter and clay were defaunated by deep freezing at 40 8C for 12 h. The materials were placed into the microcosms at field moisture content and separate samples were taken from each microcosm to determine moisture, C and nitrogen (N) contents. Moisture was measured gravimetrically after drying of the material at 90 8C for 24 h. C and N were measured using a CN analyzer (NC 2100 Soil Analyzer, Thermo Quest, Italia). The microcosms were exposed in the field for 1 or 3 years, from September 1999 to September 2002 (3-year exposure) or from September 2001 to September 2002 (1-year exposure), with six replicates for each treatment. In the 3-year exposure, 60 g dry weight equivalent of autochthonous litter prepared as above were added to each microcosm annually every autumn. The microcosms were partly buried in the fermentation and the humus layers of soil in such a way that about half of the large openings on the sides of the macrofauna-accessible microcosms were buried and half were partly above the surface. About 100 ml of the fermentation layer were placed on the top mesh of the microcosms to accelerate box inoculation by mesofauna and other smaller members of the soil biota.

J. Frouz et al. / Applied Soil Ecology 33 (2006) 308–320

2.3. Microcosm processing The litter and the mineral layers were separated after exposure. In some cases, fragmented litter formed a more or less gradual transition zone between the litter and the organic—enriched mineral layer. Thus, the net placed between the layers at the start of the experiment was used as an arbitrary border. The material above the net was assumed to belong to the litter layer and below the net to the mineral layer. Fresh weights of both litter and mineral layers were measured separately. A sample representing about half of each layer was separated, weighed again and the fauna present were extracted by Tullgren funnel for 2 days. After 2 days, the dry weight of both the litter and the fermentation layer was measured separately. Both layers were separately ground and homogenized. A 2 g sub-sample of the litter layer and 20 g of the mineral layer were powdered and prepared for C/N analysis conducted as above. Undisturbed soil samples were taken from the undried part of the mineral layer using a plastic ring, 4 cm in diameter and 8 mm high, and closed at one end by fine (0.1 mm) mesh. Undisturbed samples were carefully individually packed in plastic bags and stored in a refrigerator for later study of soil physical properties. 2.4. Microbial respiration and biomass The remainder of the undried part of the mineral layer was separately homogenized and divided into two portions. About two-third of the fresh sample was placed in a plastic bag and stored in a refrigerator until processed within 1 week, the rest was stored for membrane and storage lipid analyses by deep freezing at 40 8C. The refrigerator-stored sub-sample of the mineral layer and all of the undried litter were used to measure microbial respiration and biomass. About 3 g of the litter layer or 10 g of the mineral layer were used for these measurements. Microbial respiration was measured as CO2 production, by trapping CO2 with NaOH in an airtight vial (for 1 week in 20 8C) and subsequent titration of NaOH by HCl after BaCl addition. Microbial biomass was quantified by the chloroform fumigation–extraction method (Jenkinson and Powlson, 1976). The material was used at its original (field) moisture content for the microbial measurements. 2.5. Membrane microbial lipids To analyze membrane microbial lipids (phospholipid fatty acids, PLFA), frozen samples were thawed

311

overnight in a refrigerator at 4 8C. The lipid soluble material was extracted from 2 g of substrate as described by Bligh and Dyer (1959) and fractionated into neutral lipids, glycolipids and phospholipids on a Lichrolut column (LiChrolut Si 60, Merck, Germany) (Oravecz et al., 2004). The membrane phospholipids were subjected to a mild alkaline methanolysis (Dowling et al., 1986; Oravecz et al., 2004). The resulting fatty acid methyl esters were separated by gas chromatography (Agilent 6850, FID), using a fused silica capillary column (Ultra-2, crosslinked 5% PH ME Siloxane, 25 m, 0.2 mm, 0.33 mm; Agilent Technologies, USA). The fatty acid profiles of phospholipids (PLFA) were identified and their peak areas determined using the MIS Aerobe method of the MIDI System (Microbial ID, Inc., Newark, DE, USA). The fatty acid nomenclature follows the pattern of A:BvC, described by Frostega˚rd et al. (1993). The complete PLFA profiles were used for microbial community composition evaluation as well as for active microbial biomass estimation (Frostega˚rd, 1995). The ratio of monosaturated and saturated PLFA (MUFA/STFA ratio) was used as an indication of substrate availability for the microbial community (Bossio and Scow, 1998). Changes in PLFA characteristics for individual groups of microorganisms were used to indicate relative changes in these groups among treatments. PLFA i15:0, a15:0, 15:0, 16:1v7c, i17:0, cy17:0, cy19:0 represented bacteria (Frostega˚rd and Ba˚a˚th, 1996); 10Me18:0, 10Me17:0, 10Me16:0 actinomycetes (Kroppenstedt, 1985); 18:2v6 fungi (Frostega˚rd and Ba˚a˚th, 1996); 18:3v6, 20:4v6, 22:4v6 microeukaryota (Erwin, 1973). However, these PLFA indicators do not represent the same proportion of the total PLFA in individual microbial groups and hence cannot be used for between-group comparisons, but relative changes in these indicators between treatments can be compared. 2.6. Physical properties Undisturbed samples were used to measure water holding capacity and other hydrostatic parameters, which were derived from the retention curves. The samples were saturated and placed on the porous membrane of a suction sand tank apparatus (Klute, 1986). Suctions of 10, 30, 60 and 100 cm, respectively, were applied. For higher soil water suctions, a pressure apparatus with a ceramic membrane was used (Soil Moisture Equipment1, Santa Barbara, USA). The water holding capacity corresponds to moisture saturation, the field capacity and the wilting point are equivalent to pF 2 and 4.2, respectively (Klute, 1986). The van

312

J. Frouz et al. / Applied Soil Ecology 33 (2006) 308–320

Genuchten formula, using genetic algorithms, was applied for approximation of the retention curves (Kura´zˇ et al., 2003). 2.7. Data processing The total amount of C (or N) in individual layers was calculated as the dry weight (dw) of the layer x the C (or N) content. The dw of a layer after exposure was calculated based on the fresh weight of the whole layer and the moisture content of the Tullgren extracted subsample. The amount of C removed from the litter layer was calculated as the amount of C added in the litter layer at the start and during the experiment, minus the amount of C in the litter layer at the end of the experiment. The amount of C accumulated in the mineral layer was calculated as the amount of C in the mineral layer at the end of the experiment minus the amount of C in the mineral layer at the start of the experiment. Overall, the loss of C from the system (carbon mineralization) was calculated as the difference between C removal from the litter layer and C accumulation in the mineral layer. Values in macrofauna-acessible and non-acessible treatments were compared by t-test. Different amounts of carbon were added in reclaimed and unreclaimed site, due to different C concentrations in the autochtonous litter. Similarly, different amounts of carbon were added in the

1- and 3-year exposure treatments due to annual litter addition. To compare both sites and exposures, C removed from the litter layer, C accumulated in the mineral layer and overall loss of C were expressed as percentages of the total amounts of C introduced into the litter layer of the microcosms. A three-way ANOVA was used to compare the effects of fauna, site and duration of exposure (age). Percentage data were subject of arcsin transformation before ANOVA. Because there were two variants in each treatment, no post hoc test was needed. Because significant interactions between faunal effects and the other factors were common, the effect of fauna for individual sites and times of exposure was assessed by the t-test (N = 12). In particular, we were interesting in determining whether the effect of fauna differed between sites. So when significant interactions between fauna and site were recorded using three-way ANOVA, we applied the general linear models (GLM) to evaluate the effect of fauna separately in the two sites. We used data from both years, but year was used as a co-variate (N = 24 for GLM). In the cases of t-tests and GLM, a Bonferonni correction was applied and only p-value smaller that 0.05/n, where n is the number of tests performed in a given case (four for the t-test and two for GLM), were assumed to be significant (Sokal and Rohlf, 1981). Data manipulation was done using Microsoft Excel 7, and three-way ANOVA was conducted with SPSS 10.0.

Table 1 Carbon and nitrogen concentrations (% in soil dw; average
S.D.), in the litter and mineral layers of the microcosms, exposed in the reclaimed (R) or unreclaimed (U) sites for 1 or 3 years Litter

Mineral

C (%)

N (%)

C (%)

N (%)

R1+ R1 R3+ R3

45.63
2.19 45.22
2.79 45.11
2.31 43.47
2.38

3.38
0.26 3.28
0.29 3.15
0.33 3.02
0.28

8.04
0.72a 6.16
0.87 7.78
0.85a 5.21
0.48

0.57
0.04a 0.42
0.05 0.65
0.07a 0.49
0.05

U1+ U1 U3+ U3

36.39
0.86 41.45
4.63 42.67
1.88 38.61
5.21

1.73
0.08 1.91
0.21 1.89
0.15 1.71
0.15

7.02
0.79 6.64
0.50 19.78
0.62 17.01
0.76

0.44
0.04 0.39
0.02 0.91
0.06 0.81
0.03

Site Age Fauna Site  age Site  fauna Fauna  age

p = 0.00001 ns ns ns ns p = 0.03646

p = 0.00001 ns ns ns ns ns

p = 0.00001 p = 0.00001 p = 0.00001 p = 0.00001 p = 0.02788 ns

p = 0.00001 p = 0.00001 p = 0.00001 p = 0.00001 ns p = 0.00912

The microcosms were either accessible (+) to soil macrofauna or not (). a Marks significant difference between accessible and non-accessible treatments on the same plot and with the same length of exposure (t-test, p < 0.0125, with Bonferonni correction). Effect of site (reclaimed vs. unreclaimed), age (exposure for 1 year vs. 3 years), faunal accessibility (accessible vs. non-accessible) and their interactions were tested separately for each layer by multiple ANOVA.

J. Frouz et al. / Applied Soil Ecology 33 (2006) 308–320

3. Results 3.1. Changes in soil chemistry and physics No effect of macrofauna on C and N concentrations was observed in the litter layer. C and N litter concentrations in the unreclaimed site microcosms were significantly lower than in the reclaimed site microcosms (Table 1). In the mineral layer, macrofauna significantly affected both C and N concentrations with significant interaction between site and fauna for C only. GLM indicated that the reclaimed site C concentrations were significantly higher in the presence of macrofauna (GLM, F = 40.1, p < 0.0001), but no such significant effect was found in the unreclaimed site. Similarly, t-tests indicated that C concentrations were higher in the macrofauna-accessible microcosms in the reclaimed site, but no such significant differences were found in the unreclaimed site (Table 1). GLM indicated significantly increased N concentrations in both reclaimed and unreclaimed sites (F = 43.2 and 16.7 and p < 0.0001 and p = 0.0017 for reclaimed and unreclaimed sites, respectively). However, when individual years were compared separately by t-test, then the faunal effect appeared significant only for the reclaimed site. The C budgets in the whole microcosms and shifts of C between the individual layers are summarized in Fig. 1 and Table 2. No effect of macrofauna on C loss from the whole microcosms (both layers pooled), i.e. no effect of macrofauna on overall organic matter mineralization, was found (Fig. 1, Table 2). This can

313

be seen both from the results of three-way ANOVA using relative distribution of introduced carbon (Table 2) and the comparison of macrofauna versus no macrofauna treatments for particular sites and times using absolute values (Fig. 1). Three-way ANOVA (Table 2) indicates that access to soil macrofauna significantly increased removal of C from the litter layer and C accumulation in the mineral layer, i.e. there was a significant effect of soil fauna on soil mixing. There was, however, a significant interaction between fauna and site (Table 2). GLM showed that macrofauna significantly increased C accumulation in the mineral layer in the reclaimed site (GLM, F = 28.5, p < 0.0001) but not in the unreclaimed site ( p = 0.218). Comparison of carbon storage in the mineral layer of macrofauna-accessible and unaccessible microcosms after 3 years of exposure (Fig. 1) shows a difference of about 8 g of C per microcosm. This means that about 56 g of C per m2 was incorporated into mineral soil annually when fauna were present while in the absence of soil fauna this C would remain in litter on the soil surface. Three-way ANOVA could not be applied to hydraulic properties because the samples from the unreclaimed site after the first year of exposure were lost. However, GLM indicates significantly higher water holding capacity, field capacity and wilting point in the mineral layer for microcosms in the reclaimed site, that were accessible to macrofauna in comparison with non-accessible microcosms (GLM, F = 27.4, 31.1 and 75.4; p = 0.0001, 0.0002 and 0.0017 for water holding capacity, field capacity and wilting point,

Fig. 1. Fate of carbon introduced into the litter layer of microcosms (introduced) located in the reclaimed (R) or unreclaimed sites (U) that were exposed in the field for 1 or 3 years and were either accessible to soil macrofauna (+) or not (). Lost from litter means C lost from the litter layer, accumulated in mineral means C accumulated in the mineral layer and lost means C lost from the microcosms; asterisk (*) marks significant differences between the amount of carbon in the same pool in the microcosms from the same site and at the same age, which were either accessible for macrofauna or not (t-test, p < 0.0125 with Bonferonni correction). Bars represent S.D.

314

J. Frouz et al. / Applied Soil Ecology 33 (2006) 308–320

Table 2 Effect of site, reclaimed (R) vs. unreclaimed (U), age (1 year vs. 3 years of exposure), faunal accessibility, accessible (+) vs. non-accessible () and their interactions on the distribution of carbon added into the litter layer of microcosms on C removed from the litter layer (litter removal), C accumulated in the mineral layer and overall mineralization in the whole microcosms tested by multiple ANOVA Litter removal

Accumulation in mineral layer

Mineralization

R1+ R1 R3+ R3

49.8
12.6 39.5
5.6 73.3
7.1 a 58.5
3.8

12.0
3.9 6.3
2.5 10.7
3.4a 1.4
1.1

37.7
12.3 33.0
7.5 62.5
8.0 57.2
4.4

U1+ U1 U3+ U3

57.8
7.7 41.5
9.2 66.8
3.4 63.9
5.9

8.3
3.4 7.6
2.1 18.2
0.9 14.5
2.2

49.5
5.9 33.8
9.5 48.6
3.9 49.5
5.3

Site Age Fauna Site  age Site  fauna Fauna  age

ns p > 0.0001 p = 0.0014 ns ns ns

p > 0.0001 p > 0.0001 p > 0.0001 p > 0.0001 p = 0.0007 p = 0.0013

ns p > 0.0001 ns ns p = 0.0020 ns

Individual components of the budget are expressed as percentages of carbon introduced into the litter layer in the individual treatments. a Marks significant difference between accessible and non-accessible treatments on the same plot and with the same length of exposure (t-test, p < 0.0125, with Bonferonni correction).

respectively). Also, differences between field capacity and wilting point, which represent the amount of available water that soil can hold, were higher in macrofauna-accessible treatments in the reclaimed site (GLM, F = 15.8, p = 0.0021). Similar significant differences were shown by the t-test for the reclaimed microcosms exposed for 3 years (Table 3). No significant effects of macrofaunal accessibility on soil hydraulic properties were found in the unreclaimed site. 3.2. Effect on soil biological properties No significant effects of macrofaunal accessibility on microbial respiration or microbial biomass were

found in the litter layer of microcosms (Table 4). In the mineral layer, macrofaunal accessibility resulted in higher values of both microbial respiration and microbial biomass (Table 4). There was a significant interaction between macrofauna and site in regard to microbial biomass (Table 4). The effect of macrofaunal accessibility on microbial biomass appeared to be more pronounced in the reclaimed than in the unreclaimed site and GLM showed that macrofauna significantly increasing microbial biomass in the mineral layer in the reclaimed (GLM, F = 34.5, p = 0.00015), but not in unreclaimed site. The respiration to biomass ratio was significantly higher in the unreclaimed than in the reclaimed sites (Fig. 2), with significant interaction between fauna and site effects. In the unreclaimed site,

Table 3 Water holding capacity, field capacity and wilting point expressed as volumetric moisture (average
S.D.), in the mineral layer of the microcosms, exposed in the reclaimed (R) or unreclaimed (U) sites for 1 or 3 years Water holding capacity (%)

Field capacity (%)

Wilting point (%)

FC-WP (%)

R1+ R1 R3+ R3

41.9
2.1 34.7
1.0 43
6.8 a 23.7
2.8

27.3
4.2 24.1
0.6 33.2
2.8 a 22.7
2.6

15.1
3.3 14.7
0.6 19.4
1.2a 15.1
1.4

15.2
1.8 10.9
0.8 12.6
1.9 a 9.5
1.6

U3+ U3

15.7
1.6 26.9
5.4

13.9
1.3 20.8
5.7

9.9
1.3 12.9
3.3

4.3
0.2 8.1
2.6

The microcosms were either accessible (+) to soil macrofauna or not (). FC-WP difference between field capacity and wilting point. a Marks significant difference between accessible and non-accessible treatments on the same site in the same age (t-test, p < 0.0116 with Bonferonni correction).

J. Frouz et al. / Applied Soil Ecology 33 (2006) 308–320

315

Table 4 Microbial respiration and biomass (average
S.D.), in the litter and mineral layers of the microcosms, exposed in the reclaimed (R) or unreclaimed (U) sites for 1 or 3 years Litter

Mineral

Respiration (mg CO2 h1 g1)

Biomass (mg Cmic g1)

Respiration (mg CO2 h1 g1)

Biomass (mg Cmic g1)

R1+ R1 R3+ R3

26.0
9.5 30.1
10.8 19.3
1.9 15.6
3.6

7077.8
2945.3 15266.3
4341.2 7714.8
1619.2 7424.8
1065.7

5.2
1.8 2.6
0.8 3.4
1.6 1.5
0.5

577.2
97.4 183.7
17.3 630.9
213.1 134.8
26.5

U1+ U1 U3+ U3

28.1
4.9 36.9
4.8 30.8
1.6 27.1
3.7

8323.7
1023.1 10209.1
1753.9 7636.8
989.2 3411.7
1336.3

8.6
3.3 2.7
0.4 3.0
1.5 1.6
1.4

230.9
101.6 150.1
70.8 149.1
66.0 233.1
202.8

Site Age Fauna Site  age Site  fauna Fauna  age

p = 0.01769 p = 0.03186 ns ns ns ns

ns p = 0.01495 ns ns ns p = 0.01553

ns p = 0.01068 p = 0.00245 ns ns ns

p = 0.00785 ns p = 0.00159 ns p = 0.00151 ns

The microcosms were either accessible (+) to soil macrofauna or not (). Effect of site (reclaimed vs.unreclaimed), age (1 year vs. 3 years), faunal accessibility (accessible vs. non-accessible) and their interactions were tested separately for each layer by multiple ANOVA.

accessibility to macrofauna significantly increased the respiration/biomass ratio (GLM, F = 8.3, p = 0.0178) but no such effect of macrofauna was found in the reclaimed site. Total PLFA values, which are proportional to viable microbial biomass, were significantly higher in the reclaimed than in the unreclaimed site (Fig. 3a). When all treatments were compared together, the macrofaunaaccessible treatment showed a higher amount of total PLFA than the non-accessible treatment (Fig. 3a). The MUFA/STFA ratio, which is assumed to be an indicator of substrate availability, was higher in the presence of macrofauna when all treatments were

compared (Fig. 3b). Three-way ANOVA also showed that substrate availability was higher in the unreclaimed than in the reclaimed site. However, there were significant interactions between all three factors. When individual treatments were evaluated separately, then the effect of macrofauna was significant only in the microcosms in the unreclaimed site exposed for 3 years (t-test). PLFA markers indicate that all microbial groups increased their biomass in the mineral layer when macrofauna were present in both sites and for both exposure times (Table 5). However, this biomass increase was not the same for all groups of soil microbiota. In particular, the fungal/bacterial ratio

Fig. 2. Respiration/biomass ratio (ratio of carbon lost in microbial respiration to carbon contained in microbial biomass) in the mineral layer of microcosms located in reclaimed (R) or unreclaimed sites (U) that were exposed in the field for 1 or 3 years and were either accessible to soil macrofauna or not. Results of three-way ANOVA are presented in the inserted table. Bars represent S.D.

316

J. Frouz et al. / Applied Soil Ecology 33 (2006) 308–320

Fig. 3. Total amount of (a) PLFA and (b) MUFA/STFA ratio in the mineral layer of microcosms located in the reclaimed (R) or unreclaimed sites (U) that were exposed in the field for 1 or 3 years and were either accessible for soil macrofauna (+) or not (). Asterisk (*) marks significant differences between the microcosms in the same site and the same age, which were either accessible for macrofauna or not (t-test, p < 0.0125 with Bonferonni correction). Results of a three-way ANOVA are presented in the inserted table. Bars represent S.D.

significantly increased in treatments with macrofauna (Table 5). This effect occurred in both sites, and ANOVA did not indicate any effect of site or any interaction effect between site and fauna.

Macrofaunal accessibility had no significant effect on the density of soil mesofauna in the reclaimed site (Table 6). In the unreclaimed site mesofaunal density was considerably higher in non-accessible that in

Table 5 Amount of PLFA biomarkers (nmol g1 dw) characteristic for individual groups of microorganisms Bacteria

Fungi

Actinomycetes

Microeukaryota

F/B

R1 R1+ R3 R3+

13.10
0.90 24.75
3.90 6.94
0.42 26.88
7.95

1.07
0.06 3.09
0.99 0.43
0.11 3.11
1.05

2.00
0.07 2.62
0.14 0.84
0.02 2.40
0.75

1.44
0.22 2.68
0.76 1.18
0.29 2.41
0.92

0.08
0.01 0.12
0.02 0.06
0.02 0.12
0.01

U1 U1+ U3 U3+

9.75
0.44 11.19
1.76 6.31
0.42a 29.50
3.03

1.11
0.33 1.21
0.16 0.53
0.20a 2.55
0.29

1.29
0.19 0.86
0.15 0.76
0.02a 1.44
0.06

1.28
0.09 2.19
0.36 0.58
0.11a 2.81
0.17

0.11
0.03 0.11
0.01 0.08
0.03 0.09
0.01

Site Age Fauna Site  age Site  fauna Fauna  age

0.04297 ns 0.00001 0.00042 0.01324 ns

0.00001 0.03331 0.00057 0.00217 0.02256 0.00359

0.05092 ns 0.00001 0.03038 ns 0.02963

ns 0.04888 0.01985 ns ns 0.02334

ns ns 0.00002 ns ns ns

F/B means ratio of PLFA characteristic for fungi:PLFA characteristic for bacteria, in the mineral layer of the microcosms, exposed in the reclaimed (R) or unreclaimed (U) sites for 1 or 3 years. The microcosms were either accessible (+) to soil macrofauna or not (). a Marks significant difference between accessible and non-accessible treatment on the same site and at the same age (t-test, p < 0.0125 with Bonferonni correction). Effect of site (reclaimed vs. unreclaimed), age (1 year vs. 3 years), fauna accessibility (accessible vs. non-accessible) and their interaction were tested separately for each layer by multiple ANOVA.

J. Frouz et al. / Applied Soil Ecology 33 (2006) 308–320

317

Table 6 Numbers of Collembola and Acarina (individuals per microcosm, average
S.D.), in the litter and mineral layers of the microcosms, exposed in the reclaimed (R) or unreclaimed (U) sites for 1 or 3 years Litter

Mineral

Total

R1+ R1 R3+ R3

1240
345 1207
317 547
129 774
222

47
35 199
153 272
76 25
17

1287
351 1406
347 819
192 799
205

U1+ U1 U3+ U3

363
98 4036
572 500
35 1343
950

10
6 268
107 65
18 170
51

373
93 4304
610 565
53 1513
998

The microcosms were either accessible (+) to soil macrofauna or not (). Effect of site (reclaimed vs. unreclaimed), age (1 year vs. 3 years), fauna accessibility (accessible vs. non-accessible) and their interactions were tested separately for each layer by multiple ANOVA—no significant effect was found (but see Section 3.2).

macrofauna-accessible microcosms (Table 6). Despite the fact that these differences were large, they were only marginally significant when a Bonferonni correction was applied (t-test, p = 0.0271, 0.0157and 0.0200 for the mineral layer of microcosms with 1-year exposure, litter layer and whole microcosms with 3-year exposure, respectively). 4. Discussion Soil macrofauna did not increase significantly mineralization of organic matter (loss of organic matter from the microcosms), but accelerated the translocation of organic matter from the litter to the mineral layer (soil mixing). A preliminarily litter box experiment conducted in reclaimed sites in the same post mining area also showed no effect of macrofauna on mineralization but accelerated soil mixing and consequently C storage in the mineral layer (Frouz, 2002). However, the effect of macrofauna on soil mixing was significant only for the reclaimed site. This difference between the unreclaimed and reclaimed site may be due to differences in the composition of the macrofauna in the two sites. Long-term sampling of the same sites where the microcosms were exposed showed that earthworms, including endogeic species, were abundant in reclaimed sites (Frouz et al., 2001a; Pizˇl, 2001), while macrofauna in unreclaimed site were less abundant and dominated by millipedes and dipteran larvae (Frouz et al., 2001b, 2002). Earthworms promote mixing of the organic and mineral layers and physical binding of soil organic matter in earthworm casts (Guggenberger et al., 1996; Zhang et al., 2003). Higher earthworm abundance and consequent mixing of the organic and mineral layers may be one of the reasons for the higher C sequestration

in the mineral layer of the reclaimed site. This is supported also by earlier micromorphological observations of thin soil sections from these (Frouz et al., 2001a, 2004; Frouz and Nova´kova´, 2005). In the reclaimed site, intensive mixing of the organic and mineral layers was observed (Frouz et al., 2001a). Organic matter fragments were bound in earthworm casts and mixed into the mineral layer (Frouz et al., 2001a, 2004). By contrast, in the unreclaimed site, litter was mainly fragmented by fauna, and fecal pellets together with small particles of organic debris had accumulated at the interface between the organic and mineral layer but were not mixed into the mineral layer (Frouz et al., 2004; Frouz and Nova´kova´, 2005). In this study, we found no significant effect of fauna on overall mineralization of organic matter. This seems to be contrary to previous results obtained using litterbags either accessible or non-accessible to macrofauna, which indicated that decomposition was 5–40% higher in the presence of macrofauna (Irmer, 1995; Scheu and Wolters, 1991). However, the classical litterbag and similar enclosures imitate only the behavior of litter in the litter layer whereas our microcosms imitated more natural behavior of organic matter (long-term exposure, periodical litter additions) than classical litterbags. Moreover, the microcosms used in this study allowed us to consider the organic matter budget not only for the litter layer but also in the underlaying mineral layer. In litterbag experiments, the term decomposition is frequently used for any mass loss of litter from an enclosure. This mass loss may be caused by various processes resulting in a different final stage of organic C, such as mineralization resulting in loss of volatile CO2, leaching of water-soluble substances from the system or fragmentation of the litter and its deposition in the soil in the form of soil

318

J. Frouz et al. / Applied Soil Ecology 33 (2006) 308–320

faunal excrement. Comparison of the C content in both litter and adjacent mineral layers indicated that the processes responsible for litter decomposition and the final products differed between treatments. In treatments not accessible to macrofauna, most of the litter C loss was probably in the form of CO2 and by leaching. On the other hand, in the macrofauna-accessible treatment, the majority of the litter that was lost from the litter layer was accumulated in the mineral soil layer. Similar conclusions were made by Wachendorf et al. (1997) based on comparison of total mass loss to mass loss caused by microbial and animal respiration in litterbags. Another reason why no effect of fauna on overall mineralization was found may be that our experiment was quite long term. The processing of organic matter by soil fauna results in short term increase in microbial activity and decomposition rate. Later on, microbial activity and decomposition rates decrease and may reach lower values than in organic matter unaffected by soil fauna (Lavelle and Martin, 1992; Frouz et al., 1999). Thus, when the observation period is shorter it is more likely that a positive effect of macrofauna on decomposition rate will be found, while neutral or negative effects are more likely in long-term experiments. In agreement with other studies (Hendriksen, 1997; Frouz, 2002), accumulation of organic matter in the mineral layer resulted in higher microbial respiration and biomass. The accumulation of partly decomposed organic matter in the mineral layer may support microbial activity either by increasing nutrient availability or by modifying physical and chemical soil properties. In this study, the positive effect of soil fauna on substrate availability can be documented by the higher MUFA/STFA ratio, which is assumed to be an index of substrate availability (Bossio and Scow, 1998). It is interesting that this index of substrate availability is higher in the unreclaimed than in the reclaimed site. In the unreclaimed site, there was also higher microbial respiration relative to microbial biomass (respiration/ biomass ratio) than in the reclaimed site. A possible explanation for this finding is that the macrofauna is dominated by millipedes and dipteran larvae in the unreclaimed site (Frouz et al., 2001b, 2002). These promote litter fragmentation but not soil mixing as in earthworm dominated reclaimed sites (Frouz et al., 2001a). Litter fragmentation is likely to increase leaching of readily available nutrients into the soil, which may result in an increase in substrate availability and microbial respiration. On the other hand, earthworm mediated soil mixing results in incorporation of a

large amount of organic matter into soil, which promotes microbial biomass. Earthworms, however, support physical binding of soil organic matter in the soil (Guggenberger et al., 1996; Zhang et al., 2003). This binding of organic matter may limit nutrient availability and also microbial respiration related to microbial biomass. PLFA markers indicated an increase in all investigated groups of microflora as a result of faunal activity, which is in agreement with earlier studies focusing on the effect of gut passage on microflora (Anderson and Bignell, 1980; Krisˇtu˚fek et al., 1992; Frouz et al., 1999). PLFA indicators also showed that fungi increased relatively faster than bacteria. It is expected that this effect is doe to pH changes. Earlier measurements done at the same area (in both reclaimed and unreclaimed plots) showed that spoil material has originally an alkaline pH (8–9), but the incorporation of organic matter into this material decreased the pH to slightly acidic values (up to 5.5) (Frouz et al., 2001a; Sˇourkova´ et al., 2005). Decreased pH may favour fungi over bacteria (Blagodatskaya and Anderson, 1999; Ba˚a˚th and Anderson, 2003). As concerns physical soil properties, there was a significant increase in available water (difference between water holding capacity and wilting point) in the mineral layer of the microcosms in the reclaimed site. We expect that this increase in the ability of soil to hold water reflects earthworm activity, namely the incorporation of organic matter into the soil and physical processing of the soil. This finding is in general agreement that soil macrofauna, namely earthworms, improve soil water properties. However, empirical studies focused on the effect of soil macrofauna on water holding capacity are rare. The majority of available studies were focused on soil macrofaunal effects on porosity, aggregate stability and infiltration rate (Jegou et al., 2001; Marashi and Scullion, 2003). Some reduction of soil mesofauna densities was observed in the unreclaimed site, in microcosms accessible to soil macrofauna. Potentially, these reductions may arise from environmental modification by saprophagous macrofauna, as shown, e.g. by Lopez et al. (2003), or from predation. Because a similar effect did not occur in the reclaimed site, where modification of the soil environment by soil macrofauna was relatively more intense, this effect may correspond with access of macrofaunal predators, such as some carabid or staphylinid beetles and particularly centipedes, which are very abundant on unreclaimed site and known to be important predators of springtails (Albert, 1983; Poser, 1988).

J. Frouz et al. / Applied Soil Ecology 33 (2006) 308–320

The significant role of soil fauna in soil mixing and alteration of soil properties in the reclaimed site indicates that faster soil development in 20–30-yearold reclaimed sites, in comparison with unreclaimed sites (Frouz et al., 2004), may be at least partly due to the composition of the soil fauna. The beneficial effect of alder plantation on soil formation described by Frouz et al. (2001a) is due not only directly to the immediate effects of trees on the soil, but also indirectly to the effect of the local soil faunal community. In agreement with Stewart and Scullion (1988), we can conclude that soil fauna may play a crucial role in the reclamation process, which should not be neglected. Acknowledgements This study was supported by grants from the Czech Science Foundation (Nos. 526/01/1055, 526/03/1259), grant from the Czech Academy of Sciences (No. 1QS600660505) and Sokolovska´ uhelna´ a.s. (mining company). Research Plan of ISB AS CR No. Z6066 911. The authors thank to J. Kalcˇ´ık and T. Picek for chemical analyses and Dr. K. Edwards for reading of the manuscript. Two anonymous referees are thanked for their critical and helpful comments. References Albert, T.A.M., 1983. Characteristics of two populations of Lithobiidae (Chilopoda) determined in the laboratory and their relevance with regard to their ecological role as predators. Zool. Anz. 211, 214–225. Allison, F.E., 1973. Soil Organic Matter and Its Role in Crop Production. Elsevier, Amsterdam, New York, 637 pp. Anderson, J.M., Bignell, D.E., 1980. Bacteria in the food, gut contents and faeces of the litter feeding millipede Glomeris marginata (Villers). Soil Biol. Biochem. 12, 251–254. Anderson, J.M., Ineson, P., 1984. Interaction between microorganisms and soil invertebrates in nutrient flux pathways of forest ecosystems. In: Anderson, J.M., Rayner, A.D., Walton, D.W.H. (Eds.), Invertebrate Microbial Interactions. Cambridge University Press, Cambridge, pp. 59–88. Ba˚a˚th, E., Anderson, T.H., 2003. Comparison of soil fungal/bacterial ratios in a pH gradient using physiological and PLFA-based techniques. Soil Biol. Biochem. 35, 955–963. Blagodatskaya, E.V., Anderson, T.H., 1999. Adaptive responses of soil microbial communities under experimental acid stress in controlled laboratory studies. Appl. Soil Ecol. 11, 207–216. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Phys. 37, 911–917. Bossio, D.A., Scow, K.M., 1998. Impact of carbon and flooding on soil microbial communities: phospholipid fatty acid profiles and substrate utilization patterns. Microbial Ecol. 35, 265–278. Bradshaw, A., 1993. The reconstruction of ecosystems. J. Appl. Ecol. 20, 1–17.

319

Bradshaw, A., 1997. Restoration of mined lands—using natural processes. Ecol. Eng. 8, 255–269. Dowling, N.J.E., Widdel, F., White, D.C., 1986. Phospholipid esterlinked fatty acid biomarkers of acetate-oxidizing sulphate-reducers and other sulphide-forming bacteria. J Gen. Microbiol. 132, 1815–1825. Erwin, J.A., 1973. Fatty acids in eukarytic microorganism. In: Erwin, J.A. (Ed.), Lipids and Biomembranes of Eukaryotic Microorganisms. Academic Press, New York, pp. 41–143. ˚ ., 1995. Phospholipid fatty acid analysis to detect Frostega˚rd, A changes in soil microbial community structure. Ph.D. Thesis. Lund University, Sweden, 170 pp. ˚ ., Ba˚a˚th, E., 1996. The use of phospholipid fatty acid Frostega˚rd, A analysis to estimate bacterial and fungal biomass in soil. Biol. Fertil. Soils 22, 59–65. ˚ ., Tunlid, A., Ba˚a˚th, E., 1993. Phospholipid fatty acids Frostega˚rd, A composition, biomass, and activity of microbial communities from two soil types experimentally exposed to different heavy metals. Appl. Environ. Microbiol. 59, 3605–3617. Frouz, J., 2002. The effect of soil macrofauna on litter decomposition and soil organic matter accumulation during soil formation in spoil heaps after brown coal mining: a preliminary result. Ekologia 21, 363–369. Frouz, J., Sˇantru˚cˇkova´, H., Elhottova´, D., 1999. The effect of bibionid larvae feeding on the microbial community of litter and on reconsumed excrements. Pedobiologia 43, 221–230. Frouz, J., Keplin, B., Pizˇl, V., Tajovsky´, K., Stary´, J., Lukesˇova´, A., Nova´kova´, A., Balı´k, V., Ha´neˇl, L., Materna, J., Du¨ker, Ch., Chalupsky´, J., Rusek, J., Heinkele, T., 2001a. Soil biota and upper soil layers development in two contrasting post-mining chronosequences. Ecol. Eng. 17, 275–284. Frouz, J., Pizˇl, V., Tajovsky´, K., Balik, V., Ha´neˇl, L., Stary´, J., Lukesˇova´, A., Nova´kova´, A., 2001b. Development of soil biota in post mining forest sites established by reclamation and spontaneous succession—preliminary results of an ondoing study. In: Canizzaro, P.J. (Ed.), Proceedings of the twenty eight annual Conference on ecosystem restoration and creation. Hillsborough Community College, Tampa, Florida, pp. 122–129. Frouz, J., Pizˇl, V., Tajovsky´, K., Balı´k, V., Ha´neˇl, L., Stary´, J., Lukesˇova´, A., Nova´kova´, A., Sˇourkova´, M., Prˇikryl, I., 2002. Soil development and succession of soil biota in afforested and nonreclaimed sites in post mining landscape—preliminary results. In: Ciccu, R. (Ed.), Proceedings of Seventh International Symposium on Environmental Issues and Waste Management in Energy and Mineral Production 2002, Cagliary, Italy, pp. 621– 626. Frouz, J., Pizˇl, V., Tajovsky´, K., Balı´k, V., Stary´, J., Lukesˇova´, A., Sˇourkova´, M., 2004. The role of saprophagous macro-fauna on soil formation in reclaimed and non-reclaimed post mining sites in Central Europe. Int. J. Ecol. Environ. Sci. 30, 257–261. Frouz, J., Nova´kova´, A., 2005. Development of soil microbial properties in topsoil layer during spontaneous succession in heaps after brown coal mining in relation to humus microstructure development. Geoderma 129, 54–64. Guggenberger, G., Thomas, R.J., Zech, W., 1996. Soil organic matter within earthworm casts of an anecic-endogeic tropical pasture community, Colombia. Appl. Soil Ecol. 3, 263–274. Hendriksen, N.B., 1997. Earthworm effects on respiratory activity in a dung-soil system. Soil Biol. Biochem. 29, 347–351. Insam, H., Domsch, K.H., 1988. Relationship between soil organic carbon and microbial biomass on chronosequences of reclamation sites. Microb. Ecol. 15, 177–188.

320

J. Frouz et al. / Applied Soil Ecology 33 (2006) 308–320

Irmer, U., 1995. Die Stellung der Bodenfauna im Stoffhaushat schleswig-holstein Walder. Faunistische und Oekeologische Mitteilungen, Suppl. 18, 1–184. Jegou, D., Schrader, S., Diestel, H., Cluzeau, D., 2001. Morphological, physical and biochemical characteristics of burrow walls formed by earthworms. Appl. Soil Ecol. 17, 165–174. Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil V. A method for measuring soil biomass. Soil Biol. Biochem. 8, 209–213. Klute, A., 1986. Water retention: laboratory methods. In: Klute, A. (Ed.), Methods of Soil Analysis: Part 1. Physical and Mineralogical Methods. ASA Monograph Number, vol. 9. Soil Science Society of America, Madison, pp. 635–662. Krˇ´ıbek, B., Strnad, M., Bohacek, Z., Sykorova, I., Cejka, J., Sobalik, Z., 1998. Geochemistry of Miocene lacustrine sediments from the Sokolov Coal Basin (Czech Republic). Int. J. Coal Geol. 37, 207– 233. Krisˇtu˚fek, V., Ravasz, K., Pizˇl, V., 1992. Changes in densities of bacteria and microfungi during gut transit in Lumbricus rubellus and Aporrectodea caliginosa (Olichochaeta Lumbricidae). Soil Biol. Biochem. 24, 1499–1500. Kroppenstedt, R.M., 1985. Fatty acid and menaquinone analysis of actinomycetes and related organisms. In: Goodfellow, M., Minnikin, D.E. (Eds.), Chemical Methods in Bacterial systematics. Academic Press, London, pp. 173–199. Kura´zˇ, V., Kucˇerova´, A., Kura´zˇ, M., 2003. Using of genetic algorithm for the approximation of the retention curves (in Czech, English sum). In: Proceeding of the ‘‘Pedologicke´ dny 2003’’, Mendlova zemeˇdeˇlska´ a lesnicka´ universita v Brneˇ, pp. 107–112. Lavelle, P., Martin, A., 1992. Small-scale and large-scale effect of endogeic earthworms on soil organic matter dynamics in soil of humid tropics. Soil Biol. Biochem. 24, 1491–1498. Lavelle, P., Bignell, D., Lepage, M., Wolters, V., Rogers, P., Ineson, P., Heal, O.W., Dhillion, S., 1997. Soil function in changing world: the role of invertebrate ecosystem engineers. Eur. J. Soil Biol. 33, 159–193. Lopez, M.G., Matesanz, M.R., Lidon, J.B.J., Cosin, D.J.D., 2003. The effect of Hormogaster elisae (Hormogastridae) on the abundance of soil Collembola and Acari in laboratory cultures. Biol. Fertil. Soils 37, 231–236. Malik, A., Scullion, J., 1998. Soil development on restored opencast coal sites with particular reference to organic matter and aggregate stability. Soil Use Manage. 14, 234–239. Marashi, A.R.A., Scullion, J., 2003. Earthworm casts form stable aggregates in physically degraded soils. Biol. Fertil. Soils 37, 375– 380. Oravecz, O., Elhottova´, D., Krisˇtu˚fek, V., Sˇustr, V., Frouz, J., Trˇ´ıska, J., Ma´rialigeti, 2004. Application of Ardra and PLFA analysis in characterizing the bacterial communities of the food, gut and

excrement of saprophagous larvae of Penthetria holosericea (Diptera: Bibionidae): a pilot study. Folia Microbiol. 49, 83–93. Pizˇl, V., 2001. Earthworm succession in afforested colliery spoil heaps in the Sokolov region, Czech Republic. Restor. Ecol. 9, 359– 364. Poser, T., 1988. Chilopoden als Pra¨datoren in einem Laubwald. Pedobiologia 31, 261–281. Schafer, W.M., Nielsen, G.A., Dollhopf, D.J., Temple, K., 1979. Soil genesis, hydrological properties, root characteristics and microbial activity of 1 to 50–year old stripmine spoils. EPA-600/7-79-100. Environmental Protection Agency, Cincinnati, Ohio, 212 pp. Scheu, S., Wolters, V., 1991. Influence of fragmentation and bioturbation on the decomposition of 14C labeled beach leaf litter. Soil Biol. Biochem. 23, 1029–1034. Scullion, J., Malik, A., 2000. Earthworm activity affecting organic matter, aggregation and microbial activity in soils restored after opencast mining for coal. Soil Biol. Biochem. 32, 119–126. Scullion, J., Malinovszky, K.M., 1995. Soil factors affecting tree growth on former opencast coal land. Land Deg. Rehab. 6, 239–249. Smith, V.C., Bradford, M.A., 2003. Litter quality impacts on grassland litter decomposition are differently dependent on soil fauna across time. Appl. Soil Ecol. 24, 197–203. Sokal, R.R., Rohlf, F.J., 1981. Biometry The Principles and Practice of Statistics in Biological Research. W.H. Freeman & Co., New York, 859 pp. Sˇourkova´, M., Frouz, J., Sˇantru˚cˇkova´, H., 2005. Accumulation of carbon, nitrogen and phosphorus during soil formation on alder spoil heaps after brown-coal mining, near Sokolov (Czech Republic). Geoderma 124, 203–214. Stewart, V.I., Scullion, J., 1988. Earthworm, soil structure and the rehabilitation of former opencast coal mining land. In: Edwards, C.A., Neuhauser, E.F. (Eds.), Earthworms in Waste and Environmental Management. Academic Publishing, Hague, pp. 263–272. Stewart, V.I., Scullion, J., 1989. Principles of managing man-made soils. Soil Use Manage. 5, 109–116. Topp, W., Gemesi, O., Gru¨ning, C., Tach, P., Zhou, H., 1992. Foristische rekultivierung mit atlwaldboden in Rhenischen Braunkohlenrevier. Die suzession der bodenfauna. Zool. Jahrb. Syst. 119, 505–533. Wachendorf, C., Irmler, U., Blume, H.P., 1997. Relationships between litter fauna and chemical changes of litter during decomposition under different moisture conditions. In: Cadisch, G., Giller, K.E. (Eds.), Driven by Nature: Plant Litter Quality and Decomposition. Walingford, pp. 135–144. Zhang, X.D., Ang, J., Xie, H.T., Wang, J.K., Zech, W., 2003. Comparison of organic compounds in the particle-size fractions of earthworm casts and surrounding soil in humid Laos. Appl. Soil Ecol. 23, 147–153.