Uranium mining and milling activities adversely affect the microbial populations of impacted sites. The negative effects of uranium on soil bacteria and fungi are well studied, but little is known about the effects of radionuclides and heavy metals on archaea. The composition and diversity of archaeal communities inhabiting the waste pile of the Sliven uranium mine and the soil of the Buhovo uranium mine were investigated using 16S rRNA gene retrieval. A total of 355 archaeal clones were selected, and their 16S rDNA inserts were analysed by restriction fragment length polymorphism (RFLP) discriminating 14 different RFLP types. All evaluated archaeal 16S rRNA gene sequences belong to the 1.1b/
Metagenomic studies have revealed that Archaea are widely distributed and likely play an important role in a variety of environmental processes, such as chemoautotrophic nitrification [
Worldwide mining and milling activities have introduced high levels of radionuclides and heavy metals (HMs) into soil and aquatic environments. The adverse effects of pollutants on Archaea are not well studied [
To date, little is known concerning the interactions between archaea and U or HMs. Kashefi et al. [
The discovery that some mesophilic archaea from Crenarchaeota, which were later categorized into the new Thaumarchaeota phylum [
Intensive U mining and milling in Bulgaria were performed between 1946 and 1990 and have caused significant soil and water pollution. U production was stopped by a government decree in 1992, and mines and tailings were technically liquidated and gradually remediated. Nevertheless, their surroundings are still highly contaminated, and further contamination from the compromised remediation of mines and tailings has been recorded.
The aim of this study was to investigate the diversity of archaeal communities inhabiting environments impacted by U mining and milling activities and in particular to reveal the diversity of the archaeal
Two locations in Bulgaria were studied: the abandoned mining and milling complex “Buhovo” and the “Sliven” mine, both of which have been classified as areas of high radiological risk by the Bulgarian Agency for Radiobiology and Radioprotection. The mining complex “Buhovo” (42°45′51.20′′N; 23°34′36.86′′E) is located 30 km northeast of Sofia on a 2,280 ha territory, while the “Sliven” mine (42°41′47.68′′N; 26°22′22.47′′E) is located in South Eastern Bulgaria and occupies an area of 491 ha (Figure
Map of Bulgaria and the location of the studied sites Buhovo (BuhC and BuhD) and Sliven (Sliv).
Samples from Buhovo were collected in May 2003 at depths of 20 cm (BuhC) and 40 cm (BuhD). Samples labelled “Sliv” were collected in June 2004 from the “Sliven” mine waste pile at a depth of 40 cm. Five samples from BuhC, BuhD, and Sliv were collected under sterile conditions, transported at 4°C, and stored at −20°C until use.
The organic matter content of the sample was determined by Turyn’s method based on its oxidation by potassium dichromate [
Total DNA (>25 kb) was extracted from the samples (3 g) after direct lysis using the method described by Selenska-Pobell et al. [
Archaeal 16S rRNA genes from the genomic DNA were amplified via seminested PCR using specific archaeal
Archaeal
One archaeal and one
The sequences obtained were analysed and compared with those in the GenBank database using the BLAST server at the National Centre for Biotechnology Information (NCBI) (
The results were statistically analysed by NCSS97 (NCSS, Kaysville, Utah), and the average values were presented. The sampling efficiency and diversity within the archaeal clone libraries were estimated using the MOTHUR software program based on the furthest-neighbour algorithm, and the sequences were grouped into operational taxonomic units (OTUs) [
The sequences reported in this study were deposited in GenBank under the following accession numbers: FM897343 to FM897356 for partial archaeal 16S rRNA gene sequences and FM886822 to FM886831 for crenarchaeotic
Buhovo and Sliven samples differed in their geochemistry and the levels of U and HM contamination. BuhC and BuhD were sampled (Chromic cambisols) from different soil depths, while Sliv was a sandy gravel material collected from a mine waste pile. The texture of BuhC (20 cm at soil depth) was classified as sandy clay (35% silt and 54% clay), whereas BuhD (40 cm at soil depth) was classified as clay (38% silt and 60% clay). The bulk density of Buh soil varied in depth from 1.5-1.6 g cm−3 (20 cm) to 1.7-1.8 g cm−3 (40 cm). Soil porosity was 36–40% (20 cm) and 25–30% (40 cm) (personal communication). There is no data concerning the texture and geochemistry of Sliv substratum, except the organic matter content (0.3%) and pH (7.5). The organic matter content of the Buh samples was 2.8% for BuhC and 1.6% for BuhD. The total amount of nitrogen decreased from 1.19 g kg−1 (20 cm) to 1.03 g kg−1 (40 cm), while the total amount of phosphorus was not significantly different between the two soil layers—0.53 g kg−1 (20 cm) and 0.51 g kg−1 (40 cm). The
The main pollutants were Cu and Zn (BuhC, BuhD, and Sliv), U (BuhC and Sliv), Cr (BuhC and BuhD), As (BuhC and Sliv), Pb (Sliv), and sulfates (BuhD) (Table
Physicochemical characteristics of samples from three sites in Bulgaria polluted by uranium mining activities, expressed as means ± standard deviation (
Parameter |
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BC | BuhC | BuhD | Sliv |
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pH | — | — |
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OM | % | — |
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NO3-N | mg/kg | — |
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SO4 | mg/kg | — |
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As | mg/kg | 3.84 |
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Cd | mg/kg | 0.15 |
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Co | mg/kg | ND |
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Cr | mg/kg | 51.00 |
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Cu | mg/kg | 47.34 |
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Ni | mg/kg | 36.41 |
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Pb | mg/kg | 19.19 |
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Zn | mg/kg | 54.98 |
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U | mg/kg | 0.3–11* |
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TIAs | — | — |
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TICd | — | — |
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TICo | — | — |
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TICr | — | — |
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TICu | — | — |
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TINi | — | — |
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TIPb | — | — |
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TIZn | — | — |
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TIsum | — | — | 15.38 | 9.91 | 119.38 |
A total of 355 archaeal clones (156 from BuhC, 128 from BuhD, and 71 from Sliv) and 229
Predicted richness (Chao 1 and ACE) and diversity (Shannon-Weiner index) of BuhC, BuhD, and Sliv 16S rDNA archaeal clone libraries, expressed as means ± standard deviation.
Clone library | Number of clones | Number of OTUs | Number of singletons/doubletons | Chao 1 | ACE | Shannon-Weiner index |
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BuhCa | 156 | 7 | 4 |
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N/A |
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BuhDb | 128 | 8 | 1 |
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Slivc | 71 | 3 | 1 |
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OTUs were defined at a3%, b6%, and c9% differences in 16S rRNA gene sequences.
Rarefaction curves indicating archaeal 16S rRNA richness within BuhC (SSL 97%), BuhD (SSL 94%), and Sliv (SSL 91%) clone libraries.
The 16S rRNA gene sequences identified in BuhC, BuhD, and Sliv belonged to the 1.1b/
Phylogenetic analysis of archaeal 16S rRNA gene sequences retrieved from uranium mining sites BuhC, BuhD, and Sliv. The tree was constructed using the neighbour-joining method. The 16S rRNA sequences of
The crenarchaeotic sequences were grouped into clusters (A and B; Figure
There were common (BuhC-Ar8, BuhC-Ar18, BuhC-Ar48, and BuhD-Ar111) 16S rRNA gene archaeal sequences in the clone libraries of BuhC and BuhD. We did not retrieve any gene sequences common to the Sliv and Buh substrata.
All retrieved 16S rRNA gene sequences matched to sequences of uncultured archaea, except Sliv-Ar32, which was affiliated with the cultured archaeon
Phylogenetic analysis of 10 archaeal
Phylogenetic analysis of archaeal
All retrieved archaeal
Protein sequences derived from the same samples were also analysed, and the data validated our DNA results (data not published). The protein sequences exhibited 96–100% similarity to the closest matched GenBank sequences retrieved from terrestrial, estuarine, and hot spring environments.
The BuhC, BuhD, and Sliv archaeal communities appear to be composed solely of members of the soil-freshwater-subsurface group (1.1b) of Crenarchaeota, which was recently assigned by Bartossek et al. [
The importance of the substratum and the level of pollution in the pattern of crenarchaeotic distribution is evident from the archaeal phylogenetic tree (Figure
The distinct physical and geochemical niches of the sites harbour characteristic crenarchaeotic populations (Figure
The Buh soil environments comprise more complex and more diverse archaeal communities: 84% of OTUs and 80% of archaeal clones are from Buh, which validates data from Ochsenreiter et al. [
Archaeal diversity in Buh soil is relatively low, varying from 0.97 (BuhC) to 1.51 (BuhD), and is depth dependent. Archaeal communities of the two soil depths include both common (BuhC-Ar8, BuhC-Ar18, BuhC-Ar44, BuhC-Ar48, and BuhD-Ar111) and depth-specific 16S rRNA gene sequences, the latter of which are represented by a small number of clones (1–15 clones). The dominant OTU BuhC-Ar8 is equally distributed in soil depth, comprising 45% and 48% of clones retrieved from BuhC and BuhD, respectively. Moreover, it is closely affiliated (99% SSL) with the uncultured crenarchaeote Gitt-GR-74 (AJ535122), which is found in uranium mill tailing in Saxony, Germany [
A trend for depth dependency in archaeal distribution was also observed in other studies, which indicate that Crenarchaeota are more abundant in deeper soil layers [
The sandy gravel substratum of Sliv and its high level of pollution make this environment very unfavourable for archaeal proliferation. The inhabitants of Sliv are presented by two main OTUs (Sliv-Ar32 and Sliv-Ar22) that comprise 99% of clones. All archaeal 16S rRNA gene sequences retrieved from Sliv correspond with uncultured crenarchaeotic matches, except Sliv-Ar32, which exhibits a 99% similarity with
The phylogenetic analysis of archaeal
Forty-six percent of the archaeal
BuhC and BuhD are very different environments with regard to soil texture, nutrients, oxygen (low soil porosity), and pollution status. Nevertheless, the two environments are inhabited by ammonia-oxidizing archaea as determined by the presence of the
Phylogenetic analysis revealed that all archaeal 16S rRNA gene sequences assessed in this study belong to the 1.1b/
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was financially supported by the Institute of Resource Ecology, Helmholtz-Centre Dresden-Rossendorf, Germany.