Bacterial Diversity at Abandoned Uranium Miningand Milling Sites in Bulgaria as Revealed by 16S rRNAGenetic Diversity Study

Galina Radeva & Anelia Kenarova & Velina Bachvarova &

Katrin Flemming & Ivan Popov & Dimitar Vassilev &

Sonja Selenska-Pobell

Received: 8 May 2013 /Accepted: 10 September 2013 /Published online: 22 October 2013# Springer Science+Business Media Dordrecht 2013

Abstract Radionuclide and heavy metal contaminationinfluences the composition and diversity of bacterial com-munities, thus adversely affecting their ecological role inimpacted environments. Bacterial communities from ura-nium and heavy metal-contaminated soil environmentsandmine waste piles were analyzed using 16S rRNA generetrieval. A total of 498 clones were selected, and their 16SrDNA amplicons were analyzed by restriction fragmentlength polymorphism, which suggested a total of 220different phylotypes. The phylogenetic analysis revealedProteobacteria, Acidobacteria, and Bacteroidetes as themost common bacterial taxa for the three sites of interest.Around 20–30 % of the 16S rDNA sequences derivedfrom soil environments were identified as Proteobacteria,which increased up to 76 % (mostly Gammaproteo-bacteria) in bacterial communities inhabiting the mine

waste pile. Acidobacteria, known to be common soilinhabitants, dominated in less contaminated environ-ments, while Bacteroidetes were more abundant inhighly contaminated environments regardless of thetype of substratum (soil or excavated gravel material).Some of the sequences affiliated with Verrucomicrobia,Actinobacteria, Chloroflexi, Planctomycetes, and Candi-date division OP10 were site specific. The relationshipbetween the level of contamination and the rate ofbacterial diversity was not linear; however, the bac-terial diversity was generally higher in soil envi-ronments than in the mine waste pile. It wasconcluded that the diversity of the bacterial com-munities sampled was influenced by both the de-gree of uranium and heavy metal contamination and thesite-specific conditions.

Water Air Soil Pollut (2013) 224:1748DOI 10.1007/s11270-013-1748-1

G. Radeva :K. Flemming : S. Selenska-PobellInstitute of Resource Ecology, Helmholtz CentreDresden-Rossendorf, Bautzner Landstr. 400,01328 Dresden, Germany

K. Flemminge-mail: k.flemming@hzdr.de

S. Selenska-Pobelle-mail: s.selenska-pobell@hzdr.de

G. Radeva (*) :V. BachvarovaInstitute of Molecular Biology,Bulgarian Academy of Sciences,Acad. G. Bonchev Str., bl. 21, Sofia 1113, Bulgariae-mail: gradeva@bio21.bas.bg

V. Bachvarovae-mail: v.buchvarova@gmail.com

A. KenarovaFaculty of Biology, Sofia University, St. Kl. Ohridski,8 Dragan Tzankov blvd., 1164 Sofia, Bulgariae-mail: akenarova@abv.bg

I. PopovMolecular Medicine Center,Medical University Sofia,1 Georgy Sofiiski Str., 1431 Sofia, Bulgariae-mail: popov.bioinfo@gmail.com

D. VassilevAgrobioinstitute, 8 Dragan Tsankov blvd.,1164 Sofia, Bulgariae-mail: jim6329@gmail.com

Keywords Bacterial diversity . 16S rRNAgene . Uranium and heavy metal contamination

1 Introduction

Uranium (U) is a relatively widespread metal occurringnaturally at low levels in rocks, soil, and water (Shawkyet al. 2005). U mining and milling activities producemillions of tons of material contaminated with radionu-clides and heavy metals (HMs) (Suzuki et al. 2005). Windand water erosion of waste piles and acid mine drainageseriously contaminate natural and agricultural ecosystems,as well as aquifers, even when the mine is no longeractive (Mahmoud et al. 2005). Under such circum-stances, radionuclide and HM contamination appear tobe the main factors influencing bacterial diversity. Ra-dionuclides and HMs shift native bacterial communitiesto compositions dominated by resistant species thatappear to be tolerant to the site-specific levels ofcontamination (Vishnivetskaya et al. 2011).

A number of studies have investigated microbial com-munities in radionuclide contaminated environments usingmolecular techniques, in order to determine the phyloge-netic diversity of the associated bacterial community. De-spite the presence of contamination, which in some caseswas very high, significant complexity and diversity ofbacterial communities was observed (Fields et al. 2005;Fredrickson et al. 2004; Geissler and Selenska-Pobell2005; Geissler et al. 2009; Radeva and Selenska-Pobell2005; Satchanska and Selenska-Pobell 2005; Satchanskaet al. 2005; Suzuki et al. 2005). Most of the phyla detectedin clone libraries from these sites include groups common-ly found in natural habitats such as Proteobacteria (Akobet al. 2007; Holmes et al. 2002; Selenska-Pobell 2002),Acidobacteria (Islam et al. 2011; Mondani et al. 2011;Rastogi et al. 2010), Bacteroidetes (Akob et al. 2007;Rastogi et al. 2010), Actinobacteria (Akob et al. 2007;Barns et al. 2007; Brodie et al. 2006), Firmicutes (Akobet al. 2007; Brodie et al. 2006), and Planctomycetes (Akobet al. 2007; Brodie et al. 2006). Differences were observedin the composition of impacted bacterial communities,which were site specific and potentially connected todifferent levels of contamination and/or different geo-logical and physicochemical environmental conditions(Dhal et al. 2011).

The relative amounts of organic matter (Suzuki et al.2005), nitrates, nitrites and sulfates (Geissler and Selenska-Pobell 2005; Geissler et al. 2009), the pH (Fierer and

Jackson 2006; Nicol et al. 2008), and the redox potential(Geissler and Selenska-Pobell 2005; Geissler et al. 2009;Mondani et al. 2011) have an effect onU andHMbehaviorand toxicity. The great chemical and physical heterogene-ity of U-contaminated environments suggests selection ofdifferent U- and HM-resistant bacterial communities.

Intensive U mining and milling in Bulgaria, which wasperformed between 1946 and 1990 with an annual pro-duction of about 645 tons, has caused significant soil andwater contamination. U production was ceased by Gov-ernment decree in 1992, and mines and tailings weretechnically liquidated and gradually remediated. Neverthe-less, their surroundings are still highly contaminated, andfurther contamination from the compromised remediationof mines and tailings has been noted. The aim of this studywas to characterize and analyze the phylogenetic richnessand diversity of bacterial communities inhabitingenvironments impacted by U mining and millingactivities.

2 Materials and Methods

2.1 Sites and Sampling

Two areas in Bulgaria were studied: the abandonedmining and milling complex “Buhovo” and the“Sliven” mine, both of which have been classified asareas of high radiological risk by the Bulgarian agencyfor radiobiology and radioprotection. The mining com-plex “Buhovo” (42°45′51.20″N; 23°34′36.86″E) is lo-cated 15 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 southeastern Bulgaria and oc-cupies an area of 491 ha (Fig. 1). Mining operations atthe two sites were conducted in a conventional under-ground manner from 1962 to 1981, officially closed in1992 and remediated until 2001.

Samples from Buhovo were collected in May 2003 atdepths of 20 cm (BuhC) and 40 cm (BuhD). Sampleslabeled “Sliv” were collected in June 2004 from the“Sliven”minewaste pile at a depth of 40 cm. Five samplesfrom each site were collected under sterile conditions,transported at 4 °C, and stored at −20 °C.

2.2 Environmental Variables

The organic matter content was determined by Turyn’smethod based on its oxidation by potassium dichromate

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(Kaurichev 1980). The pH was measured using a potenti-ometer (HANA pH-meter) after the soil extract had beensuspended in 1 M KCl (soil/liquid, 1:2.5). The sulfateswere determined by spectrophotometer in 0.1 M CaCl2soil extract by the method of Bertolacini and Barney(1957). The concentration of HMs was measured usingan ELAN 5000 Inductively Coupled Plasma Mass Spec-trometer (Perkin Elmer, Shelton, CT, USA) in a 1 M HClsolution (1:20; soil/1 M HCl). The results were calculatedfor oven-dried soil.

2.3 Bacterial Abundance

Bacterial numbers were counted in 0.9 % NaCl extracts ofsoil samples (soil/NaCl, 1:100) preserved with prefilteredformaldehyde (final concentration 2 %v/v) (Fredricksonand Balkwill 1998). Subsamples (5–10 ml) were filteredthrough 0.2-μm pore-size black polycarbonate filters(Nucleopore, 25 mm diameter) and stained with acridineorange (0.01 % final concentration) according to Hobbieet al. (1977). Bacteria were counted using an epifluore-scence microscope (CETI, Belgium) and calculated pergram of oven-dried soil according to Fredrickson andBalkwill (1998).

2.4 DNA Extraction

Total DNA (>25 kb) was extracted from the samples(3 g) after direct lysis by the method of Selenska-Pobellet al. (2001), and the DNA subsamples (five DNAsubsamples per sampling site) were collected in a rep-resentative average sample for further analysis.

2.5 PCR Amplification

The 16S rRNA genes from the genomic DNAwere PCR-amplified using specific (27F) and universal (1492R) bac-terial primers (Lane 1991). Each PCR reaction mixture(20 μl) contained 200 μM deoxynucleotide triphosphates,1.25 mM MgCl2, 10 pmol DNA primers, 1–5 ng oftemplate DNA, and 1 U AmpliTaq Gold polymerase withthe corresponding 10× buffer (Perkin Elmer, Foster City,CA, USA). The PCR amplifications were done with a“touch-down” PCR in a thermal cycler Biometra(Göttingen, Germany). After an initial denaturation at94 °C for 7 min, the annealing temperature was loweredfrom 59 to 55 °C over five cycles, followed by 25 cycles at94 °C for 1min, 55 °C for 40 s, and 72 °C for 1.5min. Theamplification was completed by an extension of 20 min at72 °C.

2.6 Clone Library Construction

The 16S rDNA amplicons were cloned directly intoEscherichia coli using a TOPO TA Cloning Kit(Invitrogen, Carlsbad, CA, USA), following the manufac-turer’s instructions, to construct clone libraries for eachsample. The 16S rRNA gene inserts were subsequentlyPCR amplifiedwith plasmid-specific primers for the vectorM13 and then digested (2 h, 37 °C) with the restrictionenzymesMspI andHaeIII following the instructions of themanufacturer (Fermentas). Restriction fragment lengthpolymorphism (RFLP) patterns were visualized using3.5 % Small DNA Low Melt agarose gels (Biozym,Hessisch, Oldenburg, Germany) and then used to group

Fig. 1 Location of the studysites Buhovo (BuhC andBuhD) and Sliven (Sliv)

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clones into phylotypes. The representatives of the RFLPtypes were purified using an Edge BioSystems Quick-Step 2 PCR purification kit (MoBiTec, Gottingen, Ger-many) and then sequenced using an ABI PRISM 310DNA sequencer (Applied Biosystems, Foster City,CA, USA).

2.7 Phylogenetic Analysis

The sequences were analyzed and compared with se-quences deposited in the NCBI database (http://www.ncbi.nlm.nih.gov) using the Basic Local AlignmentSearch Tool. The presence of chimeric sequences inthe clone libraries was determined using the CHECK_CHIMERA program (http://www.wdcm.nig.ac.jp/RDP/cgis/chimera) available on the Ribosomal Data-base Project II (release 10.0) and the Bellerophonserver (http://foo.math.uq.edu.au/~huber/bellerophon.pl) (Huber et al. 2005). The sequences were alignedwith those corresponding to the closest phylogeneticrelatives using Clustal W (Thompson et al. 1994).Phylogenetic trees were constructed according to theneighbor-joining method using the Bioedit softwarepackage.

2.8 Data Analysis

The results were statistically analyzed by NCSS97(NCSS, Kaysville, UT, USA), and the average valuesare presented. The sampling efficiency and diversitywithin clone libraries were estimated using theMOTHUR software program based on the furthest-neighbor algorithm, and the sequences were groupedinto operational taxonomic units (OTUs) (Schloss et al.2009) at a sequence similarity level (SSL)≥95 % (0.05distance). For each sample, the OTU richness (rarefac-tion curves, Chao 1 and ACE) (http://putl.oclc.org/estimates) (Chao 1984) and diversity (Shannon andSimpson indices) (Magurran 1988) estimates werecalculated.

The rate of HM contamination was assessed bycalculating the sum of the technogenic coefficient ofcontamination (TCCsum) for Cd, Cu, Ni, Pb, Zn, Hg,Cr, and As whose intervention values (maximum al-lowable risk) are listed in the Bulgarian guidelines forsoil quality (Ordinance 3/2008). The TCC expressesthe ratio of the actual value to the intervention value ofeach HM, and it varies over a range of 1.0 (no need forremediation)≤TCC≥1.0 (need for remediation).

2.9 Nucleotide Sequence Accession Numbers

Sequences reported in this study were deposited inGenBank under the following accession numbers:FM866278 to FM866305 and FM877531 to FM877677for partial bacterial 16S rRNA gene sequences.

3 Results

3.1 Environmental Variables

Buhovo and Sliven samples differed in their geologicalcharacteristics and the levels of radionuclide and HMcontamination. BuhC and BuhD were sampled (Chro-mic cambisols) from different soil depths, while Slivwas a sandy gravel material collected from a minewaste pile. The texture of Buh upper soil layer(20 cm) was classified as sandy-clay (35 % silt and54 % clay), whereas that of the deeper soil layer(40 cm) was classified as clay (38 % silt and 60 %clay). The bulk density of Buh soil varied in depth from1.5–1.6 g cm−3 (20 cm) to 1.7–1.8 g cm−3 (40 cm),reflecting on soil porosity that was 36–40 % (20 cm)and 25–30 % (40 cm) (personal communication).There are no data concerning the texture and geochem-istry of Sliv substratum, except the organic mattercontent (0.3 %) and pH (7.5). The organic mattercontent of the Buh samples was: BuhC—2.8 % andBuhD—1.6 %. The total nitrogen decreased from1.19 g kg−1 (20 cm) to 1.03 g kg−1 (40 cm), while thetotal phosphorus was insignificantly different betweenthe two soil layers—0.53 g kg−1 (20 cm) and0.51 g kg−1 (40 cm). The pHH2O of BuhC and BuhDwas slightly acidic (pH 6.9 and pH 6.6, respectively).

The main contaminants were As, Zn, Cu, and Pb(BuhC, BuhD, and Sliv), U (BuhC and Sliv), Mn (BuhCand BuhD), Sr (BuhC), and sulfates (BuhD) (Table 1).Total Al in Buhovo soil was high (69,400 mg kg−1, onaverage), but it was considered to be a natural compo-nent of the soil (aluminosilicate mineral fraction) andwas probably in a biologically unavailable form consid-ering the low solubility of aluminosilicates at pH 6 to 7and the low rate of pHKCl decrease (0.1–0.5 U) incomparison to pHH2O.

According to the TCCsum, all sites were highlycontaminated and are listed in decreasing order Sliv(23.50)>BuhC (5.50)>BuhD (2.36). Moreover, thelevels of contamination might be considered as more

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serious, taking into account the Mn (BuhC and BuhD)and U (BuhC and Sliv) concentrations, which were notcalculated in the TCCsum because of the lack of Bul-garian standards.

3.2 Bacterial Abundance and Taxonomic Richness

BuhC ((8.1±2.4)×108 cell g−1) and BuhD ((7.89±1.3)×108 cell g−1) harbored high bacterial abun-dances that showed some similarities to each otherbut differed considerably (p<0.05) from Sliv ((1.65±0.6)×108 cell g−1). A total of 498 clones were selected(172 from BuhC, 176 from BuhD, and 150 from Sliv),and their 16S rDNA inserts were analyzed by RFLP,which discriminated between 220 different profiles. Theclones were grouped in 28 (BuhC), 23 (BuhD), and 22(Sliv) OTUs (Table 2), which were unique, exceptBuhC-22, which was analogous to BuhD-10.

The rarefaction curve of BuhC did not tend toward aplateau (which indicates saturation), while the curves ofBuhD and Sliv seemed to plateau (Fig. 2). Chao 1 predict-ed the highest bacterial richness in BuhC (43.6 OTUs,95 % confidence interval (CI) of 12–50), followed byBuhD (24.2 OTUs, 95 % CI of 11–33) and Sliv (22.1OTUs, 95 % CI of 12–30), as the percentage of singletons

and doubletons decreased in the same order from 59 %(BuhC) to 52 % (BuhD) and 36 % (Sliv). The ACEestimator depicted the same tendency as Chao 1. Thepredicted OTU diversity of BuhC (43.6 OTUs) was higherthan the 28 OTUs sampled, confirming the need for addi-tional sampling to completely describe the overall com-munity diversity, while that of BuhD and Slivwere close toor matched the actual OTUs sampled. At ≥95 % SSL,Chao 1 and ACE showed insignificant (p>0.05) differ-ences in bacterial richness among the sites studied.

Shannon (H′) and Simpson (D) indices showed dif-ferent trends of bacterial diversity among the sites. H′decreased in the order BuhC>BuhD>Sliv, while Dincreased in the same order (Table 2).

3.3 Bacterial Community Structures

The phylogenetic analyses based on 94 representatives ofthe predominant RFLP types and some individual RFLPsrevealed that the bacterial sequences were closely related(≥95 % SSL) to members of Proteobacteria, Acido-bacteria, Bacteroidetes, Verrucomicrobia, Gemmatimo-nadetes, Actinobacteria, Chloroflexi, Planctomycetes,Candidate division OP10, and one lineage of unclassifiedbacteria. Alphaproteobacteria (15–21 %), Betaproteo-

Table 1 Heavy metals, radionu-clide, and sulfates (in milligramsper kilogram) in Buhovo andSliven samples

ND no databValue above the maximum al-lowable concentration referringto Ordinance 3/2008aCorresponding to MPSM (2009)

Element(mg kg−1)

Backgroundconcentrationsa

Interventionconcentrations

BuhC BuhD Sliv

SO42− ND – 786 1,300 151

Al ND – 69,600 69,200 30,300

Cr 51.01 550 89.6 95.2 8.64

Mn 40.1 – 6,040 5,120 534

Fe ND – 50,800 42,800 70,000

Co ND – 29.5 27.2 22.4

Cu 47.34 500 236b 101 3,410b

Ni 36.41 300 75.2 98.4b 37.0

Zn 54.98 900 448b 464b 1,270b

As 3.84 90 274b 72.4b 412b

Sr ND – 185 118 78.4

Ag ND – 2.72 0.976 4.32

Cd 0.15 12 2.44 1.07 2.72b

Sn ND – 4.04 3.50 3.06

Hg ND 10 1.21 0.257 0.48

Pb 19.19 500 674b 126 5,160b

Th ND – 22.1 20.5 17.2

U 26.7 – 200 78.4 374

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bacteria (1–11 %), and Bacteroidetes (3–32 %) werewidespread among the sites (Fig. 3). Acidobacteria weredominant (BuhD—43 %) and subdominant (BuhC—27 %) in the Buhovo soil environments, while the domi-nant class in Sliv was Gammaproteobacteria (48 %). Theother typical sequences for Buhovo soils were those affil-iated with Verrucomicrobia and Chloroflexi.

Alphaproteobacterial sequences were grouped in threeclusters consisting of relatively equal numbers of clonesbelonging to the different sites, except cluster I, which wasdominated by the Sliv clones (16 of 27) (Fig. 4). The mostnumerous alphaproteobacterial groups were clusteredaround the sequences BuhD-23 (cluster I), BuhD-98 (clus-ter III), and Sliv-23 (cluster I), while no grouping of cloneswas recorded for BuhC (Fig. 4).

Betaproteobacterial sequences (except BuhD-322)wereretrieved mainly from Sliv (17 of 23 clones) and BuhC (5of 23 clones) (Fig. 4). The gammaproteobacterial se-quences consisted of three clusters, as clusters II (39clones) and III (29 clones) were represented only by Slivclones (Fig. 4). Deltaproteobacteria were found only inBuhD, which had the lowest contamination of HMs and Ubut the highest contamination of sulfates (Fig. 4; Table 1).

Acidobacterial sequences formed four clusters(Fig. 5). Clusters I and II were dominated by clonesfrom BuhD (17 of 22 in cluster I and 16 of 23 in clusterII), and cluster IV was a small group of bacteriaconsisting mainly of clones from BuhC (12 of 16).Cluster III was formed as a separate group based onlyon clones from Sliv (seven of seven).

Bacteroidetes sequences formed five clusters; clusters I,II, and III were dominated by sequences fromBuhC (24 of25), while clusters IVand Vwere dominated by sequencesfrom Sliv (17 of 20) (Fig. 6).Gemmatimonadetes had lowabundance in all sites studied (Fig. 3) and formed differentclusters depending on the site origin (Fig. 6). Cluster I wasconstructed by clones from Buhovo soil while cluster IIconsisted of clones from the Sliv waste pile. Some cloneswere found to represent small groups of Verrucomicrobia,Chloroflexi, Candidate division OP10 (BuhC), Bacillus(BuhC), unclassified bacteria (BuhC), Planctomycetes(BuhD and Sliv), and Actinobacteria (Sliv) (Fig. 6).

4 Discussion

Three environments with differing geological andphysicochemical characteristics, impacted by U andHM contamination, were investigated to determinetheir relative bacterial richness. Shifts in bacterialcommunities, depending on the complex effects ofU and HM loading and the site-specific environmen-tal conditions, were expected. The texture of thesubstratum differentiates the soil environments ofBuhovo from that of Sliv (a sandy gravel waste pile),while the physicochemical differences at varying soildepths differentiate the two soil layers of Buhovo asdistinct environments. The upper soil layer BuhC ismore nutrient-abundant than BuhD, but it is also morecontaminated owing to the low mobility of U and HMsand the trend of accumulation at neutral to slightly

Table 2 Statistical analysis of 16S rDNA clone libraries derived from BuhC, BuhD, and Sliv

Clone library Number of clonessequenced

Number ofOTUs

Chao1 ACE Shannon Simpson

BuhC 36 28±0.74 43.6±26.6 42.9±20 3.21±0.18 0.05±0.02

BuhD 29 23±0.43 24.2±4.6 26.3±7.0 3.00±0.16 0.06±0.01

Sliv 29 22±0.23 22.1±1.6 22.9±3.4 2.63±0.17 0.12±0.03

Diversity and predicted richness of clone libraries estimated by the Chao1 and ACE richness estimators and Shannon and Simpsondiversity indices

Fig. 2 Rarefaction analysis of BuhC, BuhD, and Sliv bacterialclone libraries at ≥95 % SSLs

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acidic pH (Bruemmer et al. 1986). In contrast to Uand HMs, sulfates penetrate deeper into the soil pro-file and accumulate in the BuhD soil layer, creating asulfur-rich environment. Sliv was the most unfavorableenvironment for bacterial growth among the sites studiedowing to its extremely low organicmatter content and high

level of contamination. The sites have been subjected tocontamination since the 1960s, providing a significantlength of time for microbial communities to adapt toelevated levels of U and HMs.

Bacterial richness among the sites decreases fromBuhCto BuD to Sliv, but the differences are not that significant,

Fig. 3 Bacterial communitycomposition of BuhC (a),BuhD (b), and Sliv (c) asrevealed by 16S rDNAlibraries

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potentially because of the high level of taxonomic identi-fication. Bacterial diversity significantly decreases (H′) or

increases (D) in the same order reflecting a decrease in thenumber of rare (H′) and an increase in the number of

Fig. 4 Neighbor-joining phylogenetic tree of proteobacterial 16S rRNA gene sequences derived from BuhC, BuhD, and Sliv withreference sequences in GenBank. The scale bar represents 0.1 substitutions per nucleotide position

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common (D) species. The above-mentioned trends in bac-terial richness are due to adaptation and selection of bac-teria under the influence of U and HM contamination aswell as the local environmental conditions. BuhD is theleast contaminated environment, but its bacterial richnessand diversity is lower than that of the more contaminatedBuhC, where the environmental conditions (organic andinorganic pool of substances) buffer the adverse effects ofcontamination (Bachmaf et al. 2008; Echevarria et al.2001; McCullough et al. 1999).

The majority of bacteria found in the three sites sam-pled are members of the Proteobacteria, Acidobacteria,and Bacteroidetes, and most of the sequences are affili-ated with matches retrieved from U- and HM-conta-minated water, sediments, and soils (Brodie et al. 2006;Geissler and Selenska-Pobell 2005; Geissler et al. 2009).Most of these taxa harbor U(VI)-reducingmembers of thegenera Geothrix, Desulfovibrio, Thiobacillus, Ferribac-terium, Geobacter, and Pseudomonas to name a few and

play a crucial role in decreasing U bioavailability andhence toxicity (Cardenas et al. 2008; Hazen and Tabak2005).

BuhC has the highest bacterial abundance and diversityamong the sites studied and is dominated by Bacteroidetes(32 %), Acidobacteria (27 %), and Proteobacteria (25 %),with low abundance species making up 15 % in total. Thedominance of Bacteroidetes is explained by their ecolog-ical importance as primary metabolizers of dead organicmatter (Elshahed et al. 2008; Thomas et al. 2011). Theresistance of some Bacteroidetes to high levels of U- andHM-contamination explains the occurrence of the classSphingobacteria in BuhC, as members of this class pro-duce sphingolipids that protect cell surfaces and functionas signaling molecules in response to a variety of environ-mental stresses (Hannun and Luberto 2000; Rickard et al.2004).

Acidobacteria are typical soil bacteria (Eichorstet al. 2007), and their contribution to the BuhC

Fig. 5 Neighbor-joining phylogenetic tree of acidobacterial 16S rRNA gene sequences derived from BuhC, BuhD, and Sliv withreference sequences in GenBank. The scale bar represents 0.1 substitutions per nucleotide position

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community is not surprising. These bacteria areproposed to be important agents in ecosystemfunctioning owing to their broad distribution, butthe insufficient data from cultivated representatives

make it difficult to ascertain their exact role in soilenvironments.

About half of the alphaproteobacterial sequencesand clones derived from BuhC are closely related to

Fig. 6 Neighbor-joining phylogenetic tree of Bacteroidetes,Gemmatimonadetes, Candidate division OP10, Verrucomicrobia,Planctomycetes, Bacillus, Actinobacteria, Chloroflexi, and unclassi-

fied bacterial 16S rRNA gene sequences derived from BuhC, BuhD,and Sliv with reference sequences in GenBank. The scale barrepresents 0.1 substitutions per nucleotide position

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plant symbionts of Rhizobiaceae, which reflects the con-tribution of leguminous plants in bacterial communitystructuring (van Spanning et al. 2005).Gemmatimonadetessequences were retrieved from all sites of interest, buthigher proportions (9 vs. 2–3 % in BuhD and Sliv) werefound in the clone library of BuhC. Gemmatimonadetesare one of the top nine phyla found in soils (Janssen 2006)and especially in arid soils suggesting an adaption to lowmoisture environments (DeBruyn et al. 2011).

The most abundant inhabitants of BuhD areAcidobacteria (43 %), Alphaproteobacteria (21 %), andVerrucomicrobia (16 %). The great share of Acidobacteriais associated with their adaptation to soil substratum andoligotrophic conditions (Eichorst et al. 2007). Acido-bacteria are abundant in BuhC (high levels of contamina-tion) and BuhD (low levels of contamination), which is ofinterest as there are contradicting opinions on their toler-ance to U and HMs. Some authors assigned Acidobacteriaas sensitive to U and HMs (Barns et al. 2007; Geissler andSelenska-Pobell 2005; Geissler et al. 2009; Rastogi et al.2010), while others (DeSantis et al. 2007; Islam and Sar2011; Mondani et al. 2011) identified this phylum in highU- and HM-contaminated environments. This, togetherwith the distribution of Acidobacteria along the soildepth from high contaminated BuhC to low contami-nated oligotrophic BuhD and the clustering ofBuhD sequences separately from that of BuhC and Sliv,implies the existence of different Acidobacteriaphylotypes that can tolerate different ranges of U andHM contamination.

Alphaproteobacteria are less abundant at BuhD, and allsequences derived from this soil layer are affiliated withuncultured bacteria. The BuhD-23 sequence, representedby five clones, is affiliated with Kaistobacter terrae(AB258386) belonging to the Sphingomonadaceae family.Nothing is known about this genus, except that some of itsmembers have been found in copper mine tailings (Pepperet al. 2012) and wastelands of copper mine tailings(JQ769532).

The distribution of Verrucomicrobia is associatedwith uncontaminated and undisturbed environments(Kielak et al. 2009). The high share of Verrucomicrobiaat BuhD (lowest contamination), absence at Sliv, andnegligible presence at BuhC (1 %) are in line withKielak et al. (2009). More unique in this case is theaffiliation of most of the Verrucomicrobia sequences (13of 14 isolated clones) with Xiphinematobacter, whichare intracellular parasites of protists and nematodes(Vandekerckhove et al. 2002).

BuhD was the only environment analyzed that wasinhabited by Deltaproteobacteria. It is assumed thatthe propagation of Deltaproteobacteria is stimulatedby the low level of oxygen (according to Schulin et al.(2004) because the soil is compact with a high level ofclay) and the high level of sulfates (1,300 mg kg−1).Some orders (Desulfobacterales, Desulfovibrionales,and Syntrophobacterales) of this class are sulfate-reducing anaerobic bacteria, some of which are foundin U-contaminated environments (Geissler andSelenska-Pobell 2005; Selenska-Pobell 2002) andHM (Villemur et al. 2006).

The Sliv bacterial community is dominated byProteobacteria (76 %), which again confirms their resis-tance to high levels of contamination (Francis et al. 2004;McLean and Beveridge 2001; Merroun et al. 2002).The most abundant Proteobacteria were theGammaproteobacteria (48 %), Alphaproteobacteria(17 %), whereas the Bataproteobacteria andBacteroidetes were less abundant both at 11 %. Thedominance of Gammaproteobacteria may be due to thecapacity of some of its members to form biofilms on stonesurfaces (Geissler and Selenska-Pobell 2005), providingpotential resistance of bacterial colonies to the toxic effectsof U and HMs, or to precipitate U as biologicallyunavailable U phosphate minerals (Merroun andSelenska-Pobell 2008; Merroun et al. 2011; Neu et al.2010). Some of the gammaproteobacterial sequences(cluster II; 39 of 39 clones) are affiliated with Legionellasp., Aquicella lusitana, and Stenotrophomonasmaltophilia, which are adapted to intracellular life inprotists and nematodes. Alphaproteobacteria are repre-sented by low numbers of sequences (one sequence; threeclones) affiliated with nitrogen-fixing bacteria and highernumbers of sequences (five sequences; 22 clones) affiliat-ed with non-nitrogen-fixing bacteria.

Most of the betaproteobacterial sequences are affil-iated with uncultured bacteria, and some of them areclosely related to rhizosphere inhabitants Ralstoniaspp. (Sliv-93; four clones), Burkholderia ferrariae(Sliv-144; two clones), and Pelomonas saccharophila(Sliv-75; two clones) (Fig. 4), which take part in nutri-ent cycling or plant pathogenesis (Álvarez et al. 2007;Gomila et al. 2007; Weber and King 2010, 2012).Burkholderia along Actinobacteria and Chloroflexiare well-known that they are among the primary colo-nists on unvegetated basalts (Dunfield and King 2004;King et al. 2008), and as vegetation cover and organiccarbon increase during biological succession, they are

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replaced by Proteobacteria (Dunfield and King 2004;Weber and King 2010).

Bacteroidetes are dominant in BuhC and subdomi-nant in Sliv, but the two sites harbor different membersof this phylum: The sequences retrieved from BuhCform clusters I–III in the phylogenetic tree, while thosefrom Sliv form clusters IV and V.

In conclusion, BuhC, BuhD, and Sliv are distinct interms of geology, physicochemistry, and levels of contam-ination. They share Alpha-, Beta-, and Gammapro-teobacteria, Acidobacteria, and Bacteroidetes in common,but also harbor site-specific bacterial taxa like Verruco-microbia and Chloroflexi (BuhC and BuhD), Plancto-mycetes (BuhC and Sliv), Deltaproteobacteria (BuhD),Bacillus, and Candidate division OP 10 (BuhC). Thebacterial communities from the three sites are similar interms of their richness, but differ significantly in theirdiversity. Both decrease from the more nutrient-abundantBuhC to BuhD to the Sliv waste pile, which was the mostunfavorable of the environments sampled. There is nolinear relationship between the level of U and HM con-tamination and the bacterial richness and diversity. Bacte-rial communities are proposed to have evolved under theinfluence of contamination, leading to selection of resistantspecies as many of the environmental sequences wereaffiliated with matches derived from U- and HM-contaminated environments. Our current investigationsare continuing with cultivation-based approaches for iso-lation, identification, and revealing the metabolic profilesof metal resistant bacteria. This study will provide a base-line data in terms of autochthonous bacterial communitiesthat could possibly serve as an indicator to gauge the rateof environmental impact of U mining and/or involve inbioremediation programs of U-polluted environments.

Acknowledgments This work was financially supported bythe National Science Fund of the Bulgarian Ministry of Educa-tion and Science (Grants 1114/04 and IFC-B-602/07) and theInstitute of Resource Ecology, HZDR, Germany.

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