INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

The presence of heavy metals in sewage sludge is often the main determinant restricting the application of sewage sludge to agricultural soil (28). After detailed assessments of the uptake and transfer of heavy metals into the food chain via crops, the Commission of the European Communities has set limits on the amount of selected heavy metals that can be added to agricultural soils receiving sewage sludge (8). However, even heavy-metal contamination that is below the upper limit set by the European Commission can have an effect on microbial community structure (1, 12, 13, 16, 32).

Many studies have focused on the effects of heavy metal on bacterial community structure (1, 16, 20, 30, 32, 34). However, no investigations have studied the effect of heavy-metal discharge on the archaean community. Until a few years ago, the domain Archaea was considered to consist only of methanogens that live under strict anoxic conditions and extremophiles that inhabit inhospitable environments (36, 39). Recent studies have shown that Archaea are also present in nonextreme environments, including marine (10, 11, 14, 27, 38a), freshwater (18), and terrestrial (3, 4, 21) ecosystems. This suggests that the Archaea also have ecological significance in nonextreme environments. Phylogenetic analysis of Archaea from nonextreme environments has revealed that they form a new cluster within the kingdom Crenarchaeota and are only distantly related to hitherto-described crenarchaea, as determined by 16S rRNA gene sequences. This group is now called the nonthermophilic Crenarchaeota and consists of at least four distinct phylogenetic clusters (4). All gene sequences isolated from soil are found in one of these clusters.

In this study, we analyzed the long-term effect of heavy metals on the archaean community in contaminated samples from five experimental field plots (Braunschweig, Germany) that received, between 1980 and 1991, different amounts of sludge and heavy metals (6). Using molecular methods such as fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE), we analyzed differences in the abundance and diversity of Archaea in both heavy-metal-contaminated soil and uncontaminated soil. A comparative sequence analysis of 16S ribosomal DNA (rDNA) libraries showed that Archaea from heavy-metal-contaminated soils cluster within the group of terrestrial nonthermophilic Crenarchaeota.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Soil characteristics and samples. Soil samples, down to a 10-cm depth, were collected at the end of November 1994 from an experimental field site in Braunschweig, Germany. The plots were characterized by the following amendments: (i) N fertilization, (ii) low sludge and low metal, (iii) low sludge and high metal, (iv) high sludge and low metal, and (v) high sludge and high metal (Table 1). All plots had been planted with spring rape. The soil, with a matrix consisting of 5% clay, 50% silt, and 45% sand, received either 100 or 300 m³ of unamended or amended sludge ha¹ year¹ between 1980 and 1991 (Table 1). The amended sludge was spiked with heavy metals (Cd, Cu, Ni, and Zn) to increase the heavy-metal load to the upper limit set by the European Commission (6). During sampling, no pronounced differences in organic C (Table 1) were noticed among these soils, although the increase in total concentrations of heavy metals Cd, Cu, Ni, and Zn from the original, noncontaminated soil to the soil with the highest metal amendment was accompanied by a decrease in pH from 7.1 to 5.3 (Table 1).

FISH. Soil samples of 5 g each were fixed in 4% paraformaldehyde-phosphate-buffered saline (0.13 M NaCl, 7 mM Na₂HPO₄, and 3 mM NaH₂PO₄ [pH 7.2] in water) on ice for 3 h (17). The suspensions were centrifuged at 4,000 × g for 5 min, and the pellets were subsequently washed with phosphate-buffered saline, resuspended in 20 ml of 96% ethanol, and stored at 20°C at a concentration of 25 mg of soil (wet weight) ml¹. Before application to slides, 40 µl of the cell suspension was dispersed in 960 µl of 0.1% sodium pyrophosphate in distilled water by mild sonication for 30 s at a setting of 8 (B-12 sonifier; Branson, Danbury, Conn.) (40). Twenty microliters was subsequently spotted onto gelatin-coated slides [0.1% gelatin, 0.01% KCr(SO₄)₂], dried at 45°C for 30 min, and finally dehydrated sequentially in 50, 80, and 96% ethanol for 3 min (each).

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DNA extraction. DNA used for PCR amplification and cloning was isolated by direct lysis of the Archaea in the soil. Ten grams of soil was homogenized for 1 min in a Waring blender with 100 ml of Crombach buffer (9). Lysis was performed according to the method of Torsvik et al. (38). Ten milliliters of the soil homogenate was lysed by the addition of 5 mg of lysozyme (Sigma Chemical Co.) ml¹. After 1 h at 37°C, 0.2 mg of proteinase K (Sigma) ml¹ was added, and the suspension was further incubated at 37°C for 0.5 h. Thereafter, SDS was added to a final concentration of 1%. The mixture was then heated to 65°C for 15 min before the lysate was cleared by centrifugation at 10,000 × g for 10 min. The lysate was purified with the Wizard total DNA cleanup system from Promega (Madison, Wisc.).

PCR. Part of the 16S rDNA was amplified from the total archaean DNA by PCR, with the GeneAmp 9600 thermocycler from Perkin-Elmer (Norwalk, Conn.). The primer sequences used for amplification were PRA46f (37) and PRU517r (22). The 100-µl reaction mixture contained the following: sterile distilled water, PCR buffer (standard 10× PCR buffer; Perkin-Elmer), 0.16% bovine serum albumin, deoxynucleoside triphosphates (each at 200 nM), primers (each at 100 µM), 2.5 U of Taq DNA polymerase (Perkin-Elmer), and template amplicon (1.9 ng). After an initial denaturation step at 95°C for 5 min, the reaction mixture was run for 30 cycles of 95°C for 0.5 min, 53°C for 0.5 min, and 72°C for 1.0 min, followed by 72°C for 6 min.

DGGE. DGGE was performed with a Hoefer SE600 gel electrophoresis unit. PCR products were loaded onto 8% acrylamide gels (bisacrylamide gel stock solution, 37:5:1; Bio-Rad Laboratories, Inc.) and run with 0.5× TAE buffer (1× TAE is 0.04 M Tris base, 0.02 M sodium acetate, and 1.0 mM EDTA [pH adjusted to 7.4]). DGGE gels contained a 20 to 60% gradient of urea-formamide solution increasing in the direction of electrophoresis. A 100% urea-formamide solution is defined as 40% (vol/vol) formamide plus 7.0 M urea. DGGE was conducted at 60°C at a voltage of 20 V for 10 min and thereafter at 200 V for 3 h. The gels were stained for 1 h with a 1:10,000 dilution of SYBR Green II (Molecular Probes, Eugene, Oreg.) in 0.5× TAE buffer before photographing.

Cloning and restriction fragment length polymorphism. PCR products from the primary PCRs were purified by preparative gel electrophoresis (1% low-melting-point NuSieve agar; FMC Bioproducts, Rockland, Maine), followed by purification with the PCR cleanup kit from Promega. The products were concentrated by precipitation by a standard procedure with sodium acetate (3 M) and 70% ethanol (31). Cloning into the pMOSBlue T vector was performed as described by the manufacturer (Amersham International Plc., Little Chalfont, Buckinghamshire, England). The clones were screened for  complementation by using X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) and IPTG (isopropyl--D-thiogalactopyranoside) (31). One hundred micrograms of ampicillin per milliliter was added to Luria agar plates (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g of agar, 1,000 ml of distilled water) for the selection of positive clones. Approximately 150 blue colonies were picked from each cloning reaction from the two soil treatments. A total of 300 blue colonies were screened for positive inserts by PCR with T7 (TAATACGACTCACTATAGGG) and U-19mer (GTTTTCCCAGTCACGACGT) primers (Amersham). PCR was performed as described above but in a total volume of 50 µl with 1.25 U of Taq DNA polymerase (Perkin-Elmer). Template was added as whole cells. The products were verified by gel electrophoresis.

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Sequence analysis. A total of 11 of the cloned amplicons with different restriction patterns and containing 16S rDNA from the low-sludge-low-metal and high-sludge-high-metal soils were sequenced. Plasmid DNA was prepared from the clones by using the Qiaprep Plasmid Miniprep kit (Qiagen, Inc., Chatsworth, Calif.). Sequencing was performed by the Advanced Biotechnology Centre (Charing Cross and Westminster Centre, London, England). The whole insert, approximately 450 bp, was sequenced by using the T7 primer as a sequencing primer. These sequences were checked for chimeric artifacts by the CHECK-CHIMERA service provided by the Ribosomal Database Project (23). Sequences were aligned with the PILEUP program of the Genetics Computer Group Software package. Distance matrices and phylogenetic trees were made by using the CLUSTAL W program. The sequences were taxonomically assigned by using the nucleotide database at GenBank and BLAST (National Center for Biotechnology Information).

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Application of DGGE revealed differences in the archaean community structure with increased heavy-metal contamination (Fig. 1). While the control soil resulted in a banding pattern of 12 bands, the low-metal soils, A and B, gave patterns of 6 bands each. However, soils amended with large amounts of heavy metals (C and D) gave banding patterns of 11 bands each. Two distinct bands (positions 5.2 and 9.6) were detected in all five plots (Fig. 1). The A and B soils showed similar banding patterns, except for one band that was unique to soil A (position 10.0) and another band that was unique to soil B (position 2.2) (Fig. 1). Likewise, the C and D soils also showed some similarities in DGGE banding patterns (Fig. 1). Three distinct bands (positions 4.3, 4.6, and 6.2) were detected only in these two plots. In addition, soils C and D each showed some faint, but unique, bands (positions 2.5, 3.1, 4.1, 8.4, and 8.9). Only one distinct band was detected in the metal-contaminated soil (position 7.4). The banding patterns from the low-sludge-low-metal plot (A) and the low-sludge-high-metal plot (B) were different from the control soil. The control, A, and B soils were somewhat similar with respect to pH (Table 1). Thus, the differences detected in the archaean community structure between the control soil and soils A and B could in part be attributed to the increase in the concentration of the heavy metals or the addition of sludge. These results were confirmed by another study showing a substantial reduction in total bacterial diversity between the control soil and soil A (low sludge-low metal) (32).

View larger version (64K): [in this window] [in a new window]   FIG. 1.   DGGE profile and schematic representations of the main bands of the DGGE gel of PCR-amplified 16S rDNA from Archaea in soil with different amendments. ctrl, soil with N fertilization; A, low sludge-low metal; B, low sludge-high metal; C, high sludge-low metal; D, high sludge-high metal.

The pHs of the A and B soils were in the same range as that of the control soil, while the pHs of the C and D soils were substantially lower (Table 1). The overall differences in the banding patterns between the control and A and B soils on one hand and the C and D soils on the other hand might therefore not be the effect of heavy metals alone but also that of an increase in the amount of sludge or a lowering in the pH (Table 1). pH has a great effect on the solubility of heavy metals (15). A twofold increase in heavy metals (Cd, Ni, and Zn) in solution, for example, has been observed after a one-unit decrease in pH (7, 33). However, differences in the banding patterns between the two low-sludge soils (A and B) and between the two high-sludge soils (C and D) must be attributed to the effect of heavy metals alone (Fig. 1).

FISH with probe ARCH915 showed that the number of Archaea belonging to this domain decreased from 1.3% ± 0.3% of the DAPI-stained cells in the control soil to numbers below the detection limit set at <1% of the DAPI-stained cells in contaminated soils (5, 40). This indicates that the heavy metals were somewhat toxic to the growth or survival of some of the Archaea. The numbers of Archaea observed in this study are in accordance with numbers found in other studies of soil ecosystems (5, 40).

Phylogenetic analysis of the Archaea 16S rDNA clone libraries revealed from 95 to 98% sequence similarity to a novel group within the kingdom Crenarchaeota (2), also known as the nonthermophilic Crenarchaeota (4). Members within this group of Archaea have recently been detected from different environmental sources (4, 18, 19, 24, 26, 27), and they were shown to be phylogenetically distinct from currently characterized crenarchaeotal species. Phylogenetic analysis of the clones from these studies has shown that the clones belong to at least four groups, which appear to have a common ancestry (4). The sequences in this study feel into three groups within the terrestrial cluster of Crenarchaeota. Two groups (clones a-195a, a-161d, a-177a, and a-176a) were similar to clones from an agricultural soil in Wisconsin (U62811 and U62814) (2), while the third group (a-9a, a-10a, a-18a, and a-3a) showed sequence similarities to clones found in freshwater sediments (U59973) (18) (Fig. 2). In another study, sequences from freshwater sediments were also shown to group within the terrestrial cluster of nonthermophilic Crenarchaeota (4).

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Neighbor-joining analysis showing the relationship of eight partial sequences from a 16S rDNA library from low-sludge-low-metal soil (with suffix "a") and high-sludge-high-metal soil (with suffix "d") to nonthermophilic Crenarchaeota 16S rDNA sequences. The FFSB cluster is a crenarchaeotal cluster from a forest soil in Finland (X96690, X96688, and X96689) (19). The marine cluster consists of the clones U78202, U78198 (24), and M88075 (10), while the terrestrial cluster contains clones from agricultural soil (U62811, U62812, U62814, U62816, and U62819) (2) and fresh water sediment (U59979 and U59973) (18). The numbers are GenBank accession numbers. Sequences determined in this study are in boldface type. Numbers at branching points show results from bootstrap analysis. The median number of changes per sequence position is shown on the scale bar.

Compared to noncontaminated soil, a significant reduction in the percentage of Archaea, as well as qualitative differences in the archaean community structure, was observed even at heavy-metal concentrations below the upper limit set by the European Commission. Molecular approaches have demonstrated that nonthermophilic Crenarchaeota are found in diverse environments and are globally distributed (2-4, 10, 18, 21, 24). Nonthermophilic Crenarchaeota have not been reported to occur in heavy-metal-contaminated soil. The findings in this study, that heavy metals were somewhat toxic to the growth or survival of some of the Archaea, may contribute to our knowledge about the ecological role of this novel group of organisms. Little is still known about their physiological characteristics and ecological significance, and further investigations are needed to fully comprehend their ecological importance.