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Soil Biology & Biochemistry

Microbial Properties of Mine Spoil Materials in the Initial Stages of Soil Development

^a Institute of Soil Science, Univ. of Halle, Weidenplan 14, 06108 Halle, Germany
^b Dep. of Crop and Soil Sciences, Pennsylvania State Univ., University Park, PA 16802
^c Dep. of Land, Air, and Water Resources, Univ. of California, One Shields Avenue, Davis, CA 95616

^* Corresponding author (mvb10{at}psu.edu)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
The early years of soil genesis during mine spoil reclamation are critical for vegetative establishment and may help predict reclamation success. Mine spoils in the Halle-Leipzig region of Germany were analyzed for microbial changes following a hay mulch-seeding treatment without topsoil or fertilizer application. Microbial biomass carbon (C_mic) and dehydrogenase activity (DHA) of spoils were measured each year in the first 3 yr after treatment. In the third year, bacterial community DNA fingerprints were compared with those from a reference soil. Microbial indicators were measured at three depths in the upper 10 cm of spoils at three sites with contrasting parent materials: glacial till (sandy loam), limnic tertiary sediments (high-lignite sandy clay loam), and quaternary sand and gravel (loamy sand). Before reclamation, C_mic means and standard deviations of surface spoils (0–1 cm) were 9 ± 6, 39 ± 11, and 38 ± 16 mg kg^–1 for the loamy sand, high-lignite sandy clay loam, and sandy loam spoils, respectively. Within one year, mean C_mic at the surface increased to 148 ± 70, 229 ± 64, and 497 ± 167 mg kg^–1, respectively, and was significantly higher at 0 to 1 cm than at lower depths. Highest DHA and DNA yields were obtained in the 0- to 1-cm depth of the sandy loam spoils. Microbial biomass C values exhibited significant correlations with DHA, DNA yield, and extractable C for all three mine spoils. Soil microbial indices were more responsive than plant measurements to differences in parent materials.

Abbreviations: C_mic, soil microbial biomass carbon • C_org, organic carbon • DHA, dehydrogenase activity • EDTA, ethylenediamine tetra-acetic acid • MineLoam1, mine spoils with sandy loam texture (Geiseltal 1 site) • MineLoam2, mine spoils with sandy clay loam texture (Geiseltal 2 site) • MineSand3, mine spoils with loamy sand texture (Goitsche site) • PCR, polymerase chain reaction • RefSoil, silt loam (from old field at Zoeberitz site) • RISA, ribosomal RNA intergenic spacer analysis • TPF, triphenylformazan • TTC, triphenyltetrazolium

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
OPEN-CAST MINE SPOIL LANDSCAPES characterize many coal-mining areas in former East Germany. In the Halle-Leipzig region, about 1.6 billion Mg of lignite were removed from open-cast mines during the last century. In 1990, programs were undertaken to amend abandoned mines and spoil banks with topsoil to reclaim these areas for agriculture or forestry. However, recreation and wildlife habitat are also common land uses in former mining areas, and these land uses typically employ reclamation treatments that do not involve addition of topsoil. Such treatments need to be assessed for their effectiveness in reclaiming mine spoils and for their suitability in meeting the needs of these land uses (Gil-Sotres et al., 1992).

Only a few studies have been conducted on reclamation of mine spoils used directly as parent material, and these have reported generally low soil microbial biomass and activity (Stroo and Jenks, 1982; Akala and Lal, 2001). Much more research has been conducted on mine spoil reclamation using amendments of municipal sludge, biosolids, or lime for subsequent agricultural land use (Fresquez et al., 1987; Insam and Domsch, 1988; Gil-Sotres et al., 1992; Sopper, 1992). A typical feature of soils derived from these spoil materials is the concentration of biomass and enzymatic activities in the upper 10 cm of the soil profile, for as long as 25 yr after reclamation (Kiss et al., 1998). The early years of soil genesis in mine spoils are critical for the establishment of a stable vegetation, and microbial biomass and activity are important indicators of soil formation (Roberts et al., 1988).

The objective of this study was to evaluate biological indices in mine spoils in the first 3 yr after a one-time reclamation treatment. The treatment consisted of slight grading, mulching with hay, and sowing with a grass-herb seed mixture (i.e., no addition of topsoil.) We measured soil microbial biomass and activity at three depths from the surface and assessed these changes in parent materials having different textures and compositions. These materials consisted of mixed tertiary and quaternary sediments with varying amounts of lignite and are typical of mine spoils that cover about 16000 ha in the Halle-Leipzig region. We expected that surface samples (0- to 1-cm depth) and medium-textured spoils would exhibit the greatest changes in microbial indices following a reclamation treatment relying on hay mulch as external nutrient source.

The relationships between soil organic matter, microbial biomass, and microbial activity have been proposed as indicators of soil maturity (Anderson and Domsch, 1990; Insam and Domsch, 1988). We measured these indicators in each of the 3 yr following reclamation. In the third year, we extracted soil microbial DNA for bacterial community fingerprinting by RISA, or ribosomal RNA intergenic spacer analysis (Johnson et al., 2003). Since little is known about whether or how the composition of soil microbial biomass influences soil development, we made qualitative comparisons of RISA fingerprints from the mine spoils with fingerprints from a local soil that had not been impacted by mining. Using a combination of microbial indices, we evaluated early changes in mine spoils after reclamation, recognizing that data collection during a longer term will be needed to confirm conclusions made in this study.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Test Sites
Three test sites were selected on the basis of contrasting spoil materials. Two test sites, Geiseltal 1 and Geiseltal 2, were located in the Geiseltal Valley, a former lignite mining district approximately 15 km southwest of the City of Halle, East Germany. The third site, Goitsche, was located about 25 km northeast of Halle. Spoil substrates for Geiseltal 1, Geiseltal 2, and Goitsche sites were glacial till, limnic tertiary sediments, and quaternary sand and gravel, respectively. Particle-size analyses by the hydrometer method (Gee and Bauder, 1986) indicated that textures of the spoil materials (0- to 10-cm depth) were sandy loam, sandy clay loam, and loamy sand, so that samples from the three sites were labeled as MineLoam1, MineLoam2, and MineSand3, respectively. Lignite mining was terminated in 1991. Reclamation began in 1993 with land surface smoothing to achieve similar slopes (7–8% or 6–7.5%) at all three sites. In the fall of 1995, replicated test plots (12.5 by 20 m) were established at the sites, all of which supported very little or no vegetation. Plots were sampled for microbial biomass at the 0- to 1-cm depth before treatment in the fall of 1995. Treatment consisted of one application of hay mulch (1 kg m^–2 mulch, or 10 Mg ha^–1), followed by sowing with a mixture of grass and herb seeds (5 g m^–2) dominated by Dactylis polygama and Melilotus alba. The hay mulch for all sites came from the same source of old field vegetation, which was dominated by native grasses. No topsoil, fertilizer, or legume inoculants were added. Therefore, the only nutrients supplied to the mine spoils were those leached from the hay mulch or released during its decomposition. By the third year of the study, all of the hay mulch residue was highly decomposed.

Surface spoils were highly heterogeneous, mainly consisting of raw tertiary and quaternary sediments with varying amounts of lignite. The mine spoils at these sites were classified as Dystri-Anthropic Regosols (ISSS/ISRIC/FAO, 1998). Before mining, surface soils at all sites had been mollisols developed from loess (Haplic Phaeozems). As an example of soil not impacted by mining, a composite sample of a local loess soil (RefSoil) was obtained in May 1998 from a single old field at the site Zoeberitz, located near Halle, midway between the Geiseltal and Goitsche sites. This old field, which had been cultivated until 1992 but kept uncultivated throughout the study period, was vegetated with densely rooted native grasses in 1998. The temperate mesic climate of the region has an annual precipitation of 460 mm and a mean annual temperature of 9°C.

Soil and Plant Sampling and Processing
Soil samples were taken from four replicate plots at Geiseltal1 (MineLoam1) and six plots at Goitsche (MineSand3). At Geiseltal2 (MineLoam2), samples were taken from six plots in 1995–1996 and four plots in 1997–1998. Estimates of plant-covered surface area in each plot were measured in each year of the study with quadrats (Braun-Blanquet, 1965). Plants in quadrats (1 by 1 m) were clipped at ground level and collected to obtain dry weights (in g m^–2) of aboveground plant material.

A total of 131 spoil bank samples at depths of 0 to 1, 1 to 5, and 5 to 10 cm was obtained in October 1996, October 1997, and May 1998. Each sample was a composite mixture of 10 soil samples. Surface samples (0–1 cm) were carefully removed with a spade, after which a stainless steel core sampler (3.5-cm diam.) was inserted into the same spot to remove the lower soil layers. Samples of about 500 g field-moist soil from each plot were sieved to obtain the <2-mm fraction and stored frozen at –18°C. Before measuring C_mic and DHA, soil samples were reconditioned by holding for 2 d at 8°C, and for 7 d at 22° to obtain a stabilized biomass (Jenkinson, 1988). Portions of the sieved fractions in October 1996 were dried for physical and chemical analysis. Additionally, in May 1998, about 20 g of composited soils from replicated mine spoil plots at each site (0- to 1- and 1- to 5-cm depths) were transferred into sterile bags, stored without sieving at –30°C for 4 mo, and thawed at 4°C before microbial DNA extraction. Composite samples of RefSoil from one field at the Zoeberitz site (no spatial replication) were obtained in May 1998 at 0- to 10- and 10- to 20-cm depths and processed in a similar manner.

Soil Physical and Chemical Analyses
Total C was determined by direct combustion to CO₂ at 950°C in a C analyzer equipped with an infrared detector. Total C estimates for MineLoam2 mine spoils included lignite as well as recent C in intimate mixture. Inorganic C was analyzed by wet oxidation using 42% phosphoric acid with evolved CO₂ detected by infrared absorption. Organic carbon (C_org) was calculated by subtracting the amount of inorganic C from total C. Total Kjeldahl N, as well as plant-available (extractable) P and K were determined with standard procedures (Hoffman et al., 1991). Values of pH were measured with a glass electrode in a paste (1 part soil, 2.5 parts 0.01 M CaCl₂ solution).

Microbial Biomass Carbon and Extractable Carbon
Microbial biomass C was determined on thawed and reconditioned samples by fumigation with alcohol-free chloroform followed by direct K₂SO₄–extraction (Vance et al., 1987). Carbon in soil extracts was analyzed with a total C_org analyzer after oxidation with Na₂S₂O₈ by UV light. Microbial biomass C was calculated by multiplying by a factor of 2.2 the difference in extractable C between the fumigated and nonfumigated samples (Wu et al., 1990). Carbon measured in K₂SO₄ extracts of nonfumigated samples was reported as extractable C (Sicora and McCoy, 1990).

Enzyme Activity
Dehydrogenase activity was determined with triphenyltetrazolium (TTC; 2,3,5-triphenyltetrazolium chloride) as electron acceptor and glucose as electron donor according to the method of Casida (1964). After mixing 1% CaCO₃ with the soil sample, 3 mL of a 1% TTC solution in water was added to 6 g soil with additional mixing, followed by static incubation for 24 h at 37°C. The concentration of alcohol-extractable triphenylformazan (TPF; alpha-phenylazo-alpha-phenylhydrazonotoluene) was measured spectrophotometrically at 520 nm. All physical, chemical, and microbiological characteristics are reported on an oven-dry weight basis (soils were dried for 24 h at 105°C).

DNA Extraction and Purification
Microbial DNA was extracted from 500-mg samples using a scaled-down version of the method of Zhou et al. (1996). A combination of cell lysis procedures (i.e., high salt, detergent, multiple freeze–thaw cycles, and chloroform lysis) was used to enhance DNA recovery. Extraction buffer (100 mM sodium phosphate [pH 8], 100 mM sodium EDTA [ethylenediamine tetra-acetic acid, pH 8], 100 mM tris-HCl [pH 8], 1.5 M NaCl, 1% CTAB [hexadecyltrimethylammonium bromide]) was prepared with a final concentration of 0.8 mg mL^–1 proteinase K. After adding 800 µL of extraction buffer, samples were vortexed for 15 s, then incubated at 37°C while shaking for 1 h. After addition of 90 µL 10% SDS, the samples were heated to 70°C for 30 min followed by freezing for 15 min. This freeze–thaw procedure was repeated twice. After the final heating, the suspension was mixed with an equal volume of chloroform-isoamyl alcohol (24:1, vol/vol) and centrifuged at low speed to separate phases. The DNA-containing aqueous phase was transferred to a clean tube. An additional volume of 600 µL tris-EDTA buffer (pH 8) was added to the chloroform-treated soil suspension for more vigorous vortexing (1 min) and after brief centrifugation, this aqueous phase was combined with the first. The pooled supernatants were treated further with chloroform-isoamyl alcohol to remove proteins. DNA was alcohol-precipitated, recovered after 30 min of centrifugation at 14000 rpm, washed with 70% ethanol, and resuspended in 10 mM tris-HCl, 1 mM EDTA, pH 7.6. The crude DNA was purified with silica gel and minicolumns, which were eluted twice with prewarmed (80°C) tris-EDTA buffer (pH 7.6). Eluted DNA was transferred to a 500-µL centrifugal filter unit for further washing and concentration. Absorbance spectra were obtained between 230 and 300 nm with a spectrophotometer to quantify DNA and assess its purity (Bruns and Buckley, 2002).

Polymerase Chain Reaction and Ribosomal RNA Intergenic Spacer Analysis
Purified soil community DNA was used as template for polymerase chain reaction (PCR) with a thermal cycler. The PCR mixture contained 1.5 mM MgCl₂, 10 mM tris-HCl (pH 9), 50 mM KCl, 200 µM of each deoxynucleoside 5'-triphosphate (dNTP), 2 U Taq DNA polymerase, and 0.4 µM each of forward primer 1406f (5'-TG(C/T) ACA CAC CGC CCG T-3') and reverse primer 115r (5'-GGC TT(T/G/C) CCC CAT CT(A/G) G-3'), in a final volume of 50 µL (Lane, 1991). These primers targeted conserved sites on bacterial 16S and 23S rRNA genes to amplify the intergenic spacer regions, which range from 200 to 1500 bp in length (Normand et al., 1996). After 4 min initial denaturation at 94°C, temperature cycling consisted of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, for a total of 30 cycles. To obtain RISA fingerprints, PCR samples were loaded in 10-µL aliquots on 5% polyacrylamide gels in 1 x tris-acetate-EDTA [20 mM tris (hydroxymethyl aminomethane), 10 mM acetate, 0.5 mM EDTA, pH 8] running buffer. Gels were subjected to electrophoresis at 150 v for 14 h, then stained for 10 min in 0.01% SYBR Green I stain (FMC BioProducts, Rockland, ME). The RISA gels were photographed, and digital images were loaded into the GelCompar 2.5 program in BioNumerics (Applied Maths, Sint-Martens-Latem, Belgium) and normalized using a 200-bp molecular weight ladder as an external reference marker. The positions of bands were determined manually using side-by-side comparisons of the fingerprint image and the densitometric curve for each lane, designating band locations at the maxima of corresponding peaks. All fingerprint comparisons were made using the same tolerance setting in BioNumerics (0.2% of the entire gel length). Species richness in each community fingerprint was estimated as the number of bands in each lane. Pairwise similarities between RISA patterns were determined using the unweighted pair-group method using arithmetic averages (UPGMA) algorithm for cluster analysis in the GelCompar program.

Statistical Analysis
Microbial biomass C and DHA data for individual depths, sites, and years following one treatment at the mine spoil sites were evaluated by repeated measures ANOVA with the GLM mixed procedure using SAS version 9 (SAS Institute, Cary, NC). RefSoil data from the Zoeberitz old field site were not included in the ANOVA, because these samples were not obtained from spatially replicated plots and could only be used as examples of a local soil that had not been mined. Microbial biomass C data from the mine spoil sites were evaluated for interactions specifying depth and year as fixed effects, and site and plot as random effects. To evaluate differences among microbial indices, Tukey's honest significant differences test was applied at P < 0.01. Pearson correlation analysis was used to compare the strength of relationships between soil properties (pH value, soil moisture, C_org content, extractable C) and C_mic for each site. Additional analyses were conducted to test relationships between C_mic, DHA, and DNA content.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Mine Spoil Properties and Soil Development
Mine spoils from the three test sites showed considerable differences in their physical and chemical properties, although no significant differences were observed in 3-yr means of plant biomass due to high variability (Table 1). MineLoam1 spoils, which consisted of glacial till (pH 7.2–7.4) with a sandy loam texture, showed highest values for plant biomass (155 g m^–2). The limnic tertiary and quaternary sediments of MineLoam2 and MineSand3 had lower values for pH (4.9–5.5) and plant biomass (86 and 114 g m^–2), respectively. MineSand3 spoils had the coarsest texture (loamy sand) and highest proportions of gravel (up to 520 g kg^–1 by weight in the 5- to 10-cm layer). The MineSand3 spoils also had the lowest water-holding capacity, as indicated by a gravimetric moisture content of 14 g kg^–1, compared with 88 and 157 g kg^–1 for MineLoam1 and MineLoam2, respectively, in 1998. The 0- to 1-cm layer of MineSand3 spoils would permit nutrients from the hay mulch to be more readily leached to lower depths than the surface layers of the other two spoils. Thus, nutrient distribution in MineSand3 Spoils would tend to be more uniform throughout the upper 10 cm of MineSand3 spoils, but lower overall.

The nutrient data in Table 1 indicated that plant establishment and growth at all three sites could have been affected by deficiencies in one or more nutrients. Plant-available P was limiting in the MineLoam1 and MineLoam2 spoils, whereas both N and K were more limiting in the MineSand3 spoils. The amounts of total N and plant-available K in MineLoam1 and MineSand3 spoils decreased continuously with depth. In contrast, N concentrations increased with depth in MineLoam2 spoils, and K concentrations did not decrease continuously or as drastically as they did in the other two spoils. These latter observations indicated that some plant-available nutrients in MineLoam2 spoils were derived from lignite-rich parent material rather than from decomposing organic matter. The poor plant productivity of MineLoam2 spoils, despite its relatively high C_org content, was consistent with findings from the study by Rumpel and Kögel-Knabner (2004), in which recent organic matter was found to be a more important nutrient source for soil biota than lignite.

Microbial Biomass
Before reclamation in the fall of 1995, samples from the 0- to 1-cm depths of MineLoam1, MineLoam2, and MineSand3 spoils had mean C_mic measurements of 38, 39, and 9 mg kg^–1, respectively. In the fall of 1996, mean C_mic measurements at 0 to 1 cm were 497, 229, and 148 mg kg^–1, respectively (Table 2). Reclamation without topsoil addition provided nutrients from decomposing hay mulch, which stimulated microbial growth during the first year. The magnitude of the C_mic increase appeared to be dependent on parent material. Microbial biomass C at the 0- to 1-cm depth of MineLoam1 spoils was significantly higher than C_mic at this same depth in either of the other two spoils (Table 2). The relatively high responsiveness of C_mic at the 0- to 1-cm depths therefore could provide early and reliable information about the suitability of parent material for revegetation.

Parent material effects on C_mic values have been demonstrated in other studies. Insam and Domsch (1988) observed that soil C_mic values ranged from 200 to 600 mg kg^–1 in loess-derived strip-mine spoils after 5 yr of crop or forest production in northwest Germany. In another study of ice-free, naturally vegetated moraine material in the arctic region of Canada, Insam and Haselwandter (1989) found that C_mic values ranged from 42 to 139 mg kg^–1. In the latter study, coarser parent material from moraine deposits could have contributed to C_mic values lower than those found for loess, although colder temperatures and less intensive plant production could also have played a role. In our study, parent material was the principal factor affecting C_mic, because all three sites were located within 40 km of each other and all were reclaimed using the same methods. On the basis of the significant interaction between depth and site for C_mic data (Table 2), our study demonstrated that parent material had an important influence on soil microorganisms and that C_mic values at 0 to 1 cm were especially responsive to parent material properties.

The ANOVA also revealed a significant interaction between site and year for C_mic data (P = 0.0274, F = 4.88, 4 df). In the first year, MineLoam1 and MineSand3 spoils had mean C_mic measurements that were significantly higher (P < 0.01) at the surface (0 to 1 cm) than at lower depths. When data for 1996 to 1998 were combined, surface C_mic measurements were significantly higher than C_mic at lower depths at all three sites (Table 2). In the second and third years, no significant changes occurred in surface C_mic at any site (Table 2). This temporal pattern, consisting of a first-year rise in microbial indices followed by several years of relatively constant measurements, was also observed for mine spoils amended with municipal sewage sludge (Sopper, 1992). Over the next 18 mo of our study, however, C_mic did decrease significantly at the two lower depths for MineLoam1 and MineLoam2 spoils (Table 2). Because of the more rapid decline in C_mic observed at the 1- to 10-cm depths, a separate measurement of C_mic at the 0- to 1-cm depth appears to be a useful tool for differentiating early soil microbial responses to parent material properties.

At all three sites in our study, C_mic decreased significantly between 0 to 1 and 1 to 5 cm, while no significant differences were observed between 1 to 5 and 5 to 10 cm (Table 2). These results differ somewhat from observations by Halvorson and Smith (1995), who tracked soil development in pyroclastic soils from volcanic ashes. In their study, C_mic declined by 50% with each 2.5- to 5.0-cm depth increment, and C_mic at the surface seemed to be correlated with the accumulation of pedogenic C_org. Higher amounts of C_org also were seen in the surface layers of MineLoam1 and MineSand3 spoils (Table 1), as well as a strong correlation between C_mic and C_org for MineSand3 soils (Table 3). Apparently, because of the high lignite content of MineLoam2 soils, the correlation between C_mic and extractable C was better than that between C_mic and C_org. A linear relationship and significant correlation (r = 0.92, P < 0.01) between C_mic and CaCl₂–extractable C also were observed by Rumpel and Kögel-Knabner (2004) in another study of reclaimed coal mine spoils. The close relationship between C_mic and extractable C appears to be based on the need for assimilable C by the soil microbial biomass (Sparling et al., 1998).

Dehydrogenase Activity
The DHA measurements of soil from 0- to 1-cm depths ranged from <1 to 230 mg TPF kg^–1 and, like C_mic, were highest in MineLoam1 spoils and lowest in MineLoam2 spoils (Fig. 1) . The DHA in the 1- to 5-cm depths also decreased in the order MineLoam1 > MineSand3 > MineLoam2 and represented only 10 to 22% of the activity in the uppermost layers. No measurable activities were observed in most samples from the 5- to 10-cm depths (Fig. 1). From 1996 to 1998, DHA remained fairly constant, with the exception of samples from MineLoam2 spoils collected in 1998, when the lowest activity at the 0- to 1-cm depth was measured. Significant linear correlation was observed between DHA and C_mic (Table 3).

View larger version (15K): [in this window] [in a new window]   Fig. 1. Dehydrogenase activities of reclaimed mine spoils and standard deviations. Bars with the same letter are not significantly different at P < 0.05.

Microbial DNA Yields from Soils
Mean microbial DNA yields ranged from 0.3 to 22.4 mg kg^–1 oven-dry soil (Table 4). The highest mean DNA yield was obtained from the 0- to 1-cm depth in the MineLoam1 spoils, and this yield was comparable with that from the upper 10 cm of RefSoil. The high DNA yield from surface samples of MineLoam1 spoils was consistent with the fact that these samples also had highest C_mic and DHA (Table 2). The DNA yields overall were congruent with C_mic data. Of the three mine spoils, only MineLoam1 exhibited significant decreases in DNA yields with depth (P < 0.01), and the difference in C_mic measured at 0- to 1- and 1- to 5-cm depths in these spoils that same year (1998) was also significant at P < 0.05. In contrast, neither DNA yields nor C_mic were significantly different with depth in the MineLoam2 and MineSand3 spoils. The lack of significant differences in DNA yield with soil depth in MineLoam2 and MineSand3 spoils appears to reflect the incipient developmental stages of these microbial communities.

Ribosomal RNA Intergenic Spacer Analysis Fingerprints
Each RISA fingerprint represented PCR products derived from 10 ng soil bacterial DNA and contained between 17 to 27 detectable bands (Table 4; Fig. 2) . The numbers of DNA bands in RISA fingerprints from each of the spoils were comparable with each other and to those observed from the uncultivated RefSoil (Table 4). Although the latter fingerprints were included as representative of a local soil not impacted by mining, they did not provide any clear information as to how band numbers and patterns might characterize a mature or successfully restored soil. The RISA fingerprint for RefSoil (0–10 cm) had the least similarity to all other fingerprints, however, and was separated on a single branch in the dendrogram shown in Fig. 2. Interestingly, the RISA fingerprint from deeper samples from RefSoil (10–20 cm) was most similar to those from MineLoam1 spoils (Fig. 2), which reinforced the interpretation that MineLoam1 spoils provide more favorable conditions than the other two spoils for microbial community development.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Community similarities based on RISA (ribosomal RNA intergenic spacer analysis) gel band patterns: Dendrogram showing similarities among soil communities and gel band patterns for reclaimed mine soils at 0- to 1- and 1- to 5-cm depths.

Pairwise similarities were lowest for 0- to 10- and 10- to 20-cm depths of the RefSoil (0.27). Community divergence at greater depth has been observed in other studies (Felske and Akkermans, 1998; Fierer et al., 2003; Agnelli et al., 2004) and appears to reflect microbial community response to soil physicochemical heterogeneity during longer periods of residence time. A contrasting example was described by Boon et al. (2000), who found that the similarities of 16S rRNA gene sequences derived from samples at different depths in a landfill sediment were always higher than 0.90. In their study, Boon and coworkers emphasized that the physical and chemical properties of their soils were homogeneously distributed with depth. This homogeneity apparently reflected the consistency of microbial community patterns. Considering the short time period after reclamation in our study, it appears that more time must elapse before the microbial community structures in mine spoils become spatially differentiated with respect to soil depth.

RISA interpretation is confounded by the fact that fingerprint bands can arise from PCR amplification of DNA from dormant or inactive microbial populations (Bruns and Buckley, 2002), the provenance of which would likely be different in young and mature soils. Additionally, RISA fingerprints in our study were obtained from composited soil samples, which would further interfere with interpretations based on spatial and physical differences. More informative molecular techniques would be needed to characterize microbial communities which reflect a soil's recovered productivity. Ideally, such techniques would involve analysis of microbial RNA, rather than DNA, inclusion of fungal populations, and evaluation of greater numbers of samples in spatially controlled experimental designs.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Our study documented significant increases in C_mic at the surfaces of tertiary and quaternary sedimentary parent materials within 1 yr of a reclamation treatment that did not involve topsoil addition or sludge application. The highest C_mic at all sites was measured at 0- to 1-cm depths, where easily decomposable sources of organic matter would be concentrated. Microbial biomass C and DHA values measured below 0 to 1 cm, however, declined significantly in the MineLoam1 and MineLoam2 spoils within 3 yr following reclamation, while values for the MineSand3 spoils were low overall. Raw parent material properties affected microbial indices of reclaimed mine spoils in predictable ways. Microbial biomass C was lowest in the MineSand3 spoils, which had the coarsest texture and lowest pH, and highest in the MineLoam1 spoils, which had medium texture and more neutral pH. The decline in microbial indices during the 3-yr period indicated that plant C inputs were not sufficient to sustain the increased microbial biomass that had become established immediately after the mulching and seeding treatment. The implications for reclamation management are that microbial habitat quality is intimately tied to the suitability of mine spoils as plant habitat. Soil microbial indices appeared to be more informative than plant responses about the early effects of reclamation, because microbial indicators responded more clearly to parent material properties than did plant density measurements, which were highly variable. In the absence of a clear indicator soil to use as a target for restoration assessment, the main criterion for early reclamation success in our study was the demonstration of a significant increase in microbial biomass at the given site. The microbial response observed in the most favorable parent material of the three spoils (MineLoam1) could be considered to represent a target response level that might be achievable with other mine spoils with less favorable properties. Results of initial chemical and physical measurements of mine spoil materials could therefore be used to guide the selection of additional reclamation treatments, such as lime, fertilizers, or higher mulch applications, for droughty or acidic spoils.

Significant correlations between C_mic and DHA and extractable C indicate their potential utility as rapid assessment tools for mine spoil reclamation. Differences in DNA yields and RISA fingerprints also were consistent with changes in other microbial indices, although more informative molecular techniques targeting active populations of fungi as well as bacteria would be more appropriate. Molecular assays could thus complement process-based measurements in evaluating restoration progress in anthropogenic soils.

	ACKNOWLEDGMENTS

 
The authors thank Professor Marvin Risius for statistical analysis advice.

Received for publication August 13, 2004.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

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