Journal of Environmental Science and Health, Part A (2014) 49, 694–709 C Taylor & Francis Group, LLC Copyright  ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2014.865458

Microbial communities in uranium mine tailings and mine water sediment from Jaduguda U mine, India: A culture independent analysis PALTU KUMAR DHAL1,2 and PINAKI SAR1 1

Department of Biotechnology, Indian Institute of Technology Kharagpur, India Biomedical Informatics Center, National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, West Bengal, India

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Microbial diversity within uranium mine tailings and mine water sediment from the Jaduguda uranium mine, India was characterized by metagenome-derived 16S rRNA gene clone libraries. Samples from fresh tailings (JFT244), abandoned (vegetated) tailings (JOT245) and mine water sediment (J1-5) having wide ranges of pH (5.7 to 10.4), nitrogen, phosphorus and organic carbon [150–5700 ppm, 800–9100 ppm and 0.18–6.5% (w/w)] and elevated metals (Ni, Cu, Zn and U) were used to explore the inhabitant bacterial and archaeal community structures. Consistent to the sample’s physicochemical properties, up to four orders of magnitude variation in bacterial CFU counts was observed. The data showed that with increasing metal and decreasing nutrient (organic C, N, P, etc.) contents, microbial diversity indices decrease within the samples. Culture-independent analyses revealed predominance of phyla Proteobacteria and/or Acidobacteria within the samples along with members of Actinobacteria, Cyanobacteria, Chloroflexi, Genera incertae sedis OP10, Firmicutes and Planctomycete as relatively minor groups. Abundance of Crenarchaeota in tailings samples and Euryachaeota in mine water sediment was noted. Diversity of dissimilatory sulfate reductase gene (dsr) was studied. Putative metabolic properties as derived from taxonomy and phylogenetic lineages indicated presence of chemolithotrophic and heteotrophic aerobic and anaerobic organisms capable of nitrogen fixation, nitrate reduction and biogeochemical cycling of metals, sulfur and methane. The data indicated that indigenous microbial populations are capable of maintaining self-sustenance in these highly hazardous environments and possess catalytic potential for their use in in situ bioremediation. Keywords: Uranium mine, microbial diversity, metagenome, tailings, 16S rRNA gene, dissimilatory sulfate reductase, bioremediation.

Introduction Controlled discharge of tailings and other process effluents generated during extraction and beneficiation of U ores represents one of the major burdens of anthropogenic environmental radioactivity.[1] The nature of U tailings is unique, due to its significant radioactivity contributed mostly by decay products of U with long half-lives (e.g., Th230: 75,000 years; Ra226: 1,600 years).[2] Additionally, like other tailings, U mine tailings also contain reduced organic matters and essential nutrients like nitrogen and phosphorous, but elevated concentration (1 to 50 g/kg) of multiple metals (As, Cu, Fe, Mn, Ni, Pb, Cd and Zn) and often devoid of vegetation.[3] The global impact of such waste is enormous, as unreclaimed sites mostly remain unvegetated Address correspondence to Pinaki Sar, Department of Biotechnology, Indian Institute of Technology, Kharagpur, 721302, India; E-mail: [email protected]; [email protected] Received June 21, 2013. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lesa.

for long periods (tens to hundreds of years), and exposed tailings can spread over larger areas via dispersion and erosion.[3] Off-site migration of contaminants from these large-volume hazardous waste sites is of severe environmental concern and therefore understanding factors that regulate environmental fate of these contaminants and their migration strategies has generated enormous scientific interest.[4-6] It has been proven conclusively that microorganisms play defining roles in controlling the speciation and mobility of a wide range of metals and radionuclides in engineered and natural environments.[5,7,8] Hence, understanding the microbial community composition and function in in situ biogeochemical transformation of contaminants is considered imperative for developing long term stabilization strategies for tailings and other similar wastes. Particularly, due to the geochemical complexities at radionuclide- and metalcontaminated mixed waste sites that make their remediation a complex process with significant economical and technological challenges, in situ bioremediation with microorganisms remains potentially the most suitable, costeffective and viable cleanup technology.[7,8]

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Microbial communities in uranium mine tailings In the last few years microbial community composition and function within metal/radionuclide contaminated environments including those impacted by U mines have been extensively studied using culture-independent and dependent methods [8-12] and many others). All these studies have demonstrated that uranium-contaminated sites harbour metabolically diverse microorganisms. They are capable of executing various modes of metal–microbe interaction to maintain viability and can dramatically impact the form and distribution of metal influencing the long-term geochemistry of such sites.[6,8,13] Although it has become increasingly clear that certain microbial activities catalyzed by specific populations may be responsible for such process (reducing environmental toxicity and availability of metals), nature of microbial groups and mechanisms responsible for these process under the prevailing conditions relevant for natural attenuation of contaminated sites remain unclear.[6] Compared to several studies reporting the microbial diversity in U-contaminated environments, only few investigations were conducted on actual uranium mine wastes, particularly mine tailings.[14,15] Except the very recent report by Bondici et al.[13] culture-independent analysis of microbial diversity in U mine tailings sites remains unexplored and uncharacterized. The U mine at Jaduguda is the oldest operating mine in India (since 1960). The milling plant receives ores from Jaduguda and several adjoining U-mines and has the capacity to process about 2100 tones ores per day leading to the discharge of around 1.5 × 105 m3 of solid waste to the tailings pond per annum. It has been estimated that apart from the metallic co-contaminants present within this large volume of tailings, Jaduguda mine tailings also pose tremendous radioactive hazard with γ -radiation dose rates of 0.8 to 3.3 mGy h−1. The geometric mean activity concentrations of 222Rn in air over the fresh and abandoned (vegetated) tailings ponds were found to be 30 and 23 Bqm−3, respectively.[16] Microbial diversity within U ores and mine adjoining sites at Jaduguda and other nearby U mines, along with remediation potential of indigenous bacterial strains from those sites have been previously investigated by us.[11,17–19] During the present study we have characterize the microbial community structure and composition at U mine tailings and mine water sediment; the two most important waste sites of U mines. Along with characterizing site’s physicochemical parameters, bacterial and archebacterial diversity was explored studied using culture-independent methods. Attempts were made to elucidate possible correlation between geochemical properties of these samples and their inhabitant microbial communities. Diversity of dissimilatory sulfate reductase gene (dsr), (involved in anaerobic sulfate reduction by sulfate-reducing bacteria) known as an important players in microbial reduction, and microbial precipitation of metals and radionuclides was ascertained from community metagenomes.

Material and methods Sampling sites and sample collection Uranium mine tailings and mine water sediment samples were collected from Jaduguda U mine (Table 1). At east Singbhum district of Jaduguda (known as Singhbhum Thrust Belt of Jharkhand State) there are several U mines located at Jaduguda, Bhatin, and Bagjata, Turamdih and Narwapahar. Uranium ore from many of these mines are brought to the Jaduguda milling plant and processed; leading to the generation of large quantity of highly hazardous tailings deposited within engineered ponds, referred to as “tailings ponds.” Uranium tailings ponds at Jaduguda are surrounded by hills on the three sides and an engineered dam on the downstream side. Locations of these ponds are almost in the middle of Shinghbhum shear zone and are nearly 2 km away from the mill.[20] At the tailings pond, settling of solid takes place slowly and the supernatant liquid effluent is decanted out for further removal of dissolved metals and radionuclides before its discharged to nearby Juria-Gara-Suvarnrekha river ecosystem.[21] There are three tailings ponds at Jaduguda, named as TP-1, TP-2 and TP-3. TP-1 and TP-2 are saturated in operations [20] and act as abandoned ponds while TP-3 is the active pond and fresh dumping of tailings is being carried out there. In the present study, TP1 is referred as vegetated tailings since the top of the tailings pond is now covered by vegetation of different grasses, while TP-3 is referred to as fresh tailings. Samples were collected from tailings ponds TP-1 and TP-3 during July 2006 to September 2007. In addition to the two tailings, a third sample was collected from mine water tank, that stores circulating mine water. For TP-3, samples were collected directly from the outlet of feeding pipes discharging fresh tailings within the pond. For TP1, sediment samples were obtained from nearly 10–15 cm depth. Samples collected from TP-1 and TP-3 were designed as JFT245 and JOT244, respectively. From mine water tank, the sediment settled below the pit was obtained and designed as J1-5. All samples were collected aseptically and stored immediately in ice until further analysis. Physicochemical and microbiological analysis Physiochemical parameters including pH, dissolved oxygen (D.O), conductivity and salinity of the samples were measured during sample collection using Orion star series multi parameter (Thermo Orion meter, Beverly, MA, USA). Heavy metals and actinide elements were estimated by inductively coupled plasma mass spectrometry (ICPMS) (Varian, Palo Alto, CA, USA) and/or atomic absorption spectroscopy (AAS) (Perkin Elmer, Waltham, MA, USA). Total organic carbon (TOC), total nitrogen (TN),

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Table 1. Geomicrobial characteristics of the samples. Samples

Nature of Sample GPS Data Temp (◦ C) D.O (mg L−1) pH Conductivity (µs cm−1) Total organic carbon (%) Phosphorous (ppm) Sulfur (ppm) Potassium (ppm) Nitrogen (ppm) Moisture (%) Metals (ppm) Cr Co Ni Cu Zn Cd Th U

JFT244

JOT245

J1-5

Fresh tailings influent (TP-III) N 22◦ 39.085 E 86◦ 19.800 34.3 ± 0.01 6.9 ± 0.001 10.43 ± 0.01 4340 ± 10.2 0.18 ± 0.002 800 ± 4.2 25 ± 0.01 400 ± 9.01 150 ± 2.01 89.5 ± 4.2

Vegetative tailing pond (TP-I)

Mine sediment

N 22◦ 39.489 E 86◦ 20.216 34.1 ± 0.01 2.9 ± 0.001 5.75 ± 0.01 4020 ± 10.8 0.73 ± 0.02 6300 ± 10.2 135.6 ± 5.01 5200 ± 11.1 5700 ± 19.1 30.16 ± 2.1

79.4 ± 1.2 91.8 ± 2.3 1267.7 ± 4.2 494 ± 4.1 624.1 ± 4.2 3.1 ± 0.2 17.5 ± 1.1 90 ± 1.1

6.3 ± 2.0 5.2 ± 0.9 102.5 ± 0.2 306.7 ± 7.2 338.8 ± 3.5 2.4 ± 0.1 5.7 ± 0.7 4.3 ± 0.2

Microbial counts (per ml/gm of sample) R2A (pH-7.0) 4 × 105 ± 0.9 PTYG (pH-7.0) 1 × 104 ± 1.1 MGY (pH-3.0) 3 × 103 ± 0.7

1 × 108 ± 0.6 1 × 108 ± 0.2 6 × 101 ± 1.9

Eubacterial 16S rRNA gene clone library Number of clones Richness Simpsons (1/D) Shannon (H) Equitability (E)

139 51 1.04 3.51 0.89

Archaebacterial 16S rRNA gene clone library Number of clones 65 Richness 6 Simpsons (1/D) 2.28 Shannon (H) 0.96 Equitability (E) 0.53 R2A (pH-7.0) 4 × 105 ± 0.9

total phosphorous (TP) and total sulfur (TS) were analyzed by standard procedures.[22] For enumeration of bacterial counts, 5 g or 5 mL of each sample was resuspended in sterile saline (45 mL), mixed thoroughly in a rotory shaker (200 rpm, 1 h) and 100 µl of suspension was plated on different media following serial dilution. Enumeration of culturable bacterial populations was estimated in agar plates prepared with the following media: R2A, PTYG and MGY.[23] Upon inoculation, plates were incubated at 30◦ C in dark

153 54 1.03 3.69 0.92 58 11 1.13 2.25 0.93 1 × 108 ± 0.6

N 22◦ 39.099 E 86◦ 20.778 34.4 ± 0.01 2.4 ± 0.001 7.3 ± 0.01 950 ± 6.2 6.5 ± 1.2 9100 ± 17.2 585 ± 9.2 7100 ± 14.2 5500 ± 10.1 78.9 ± 3.7 2.7 ± 0.1 31.9 ± 1.1 1749 ± 11.2 174 ± 1.2 107.4 ± 5.2 2.1 ± 0.1 6 ± 0.6 30 ± 0.2 1 × 109 ± − 0.5 8.5 × 107 ± 1.8 5 × 103 ± 2 71 57 1.0 3.96 0.97 77 8 1.38 1.7 0.82 1 × 109 ± 0.5

and examined over a period of 2 weeks and numbers of colonies were counted. Extraction of community DNA and PCR amplification of 16S rRNA and dsr genes Total bacterial community DNA from all these samples were extracted using Power soilTM DNA kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA). Bacterial and

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Microbial communities in uranium mine tailings archebacterial 16S rRNA genes were amplified from the pooled DNA samples (of triplicate extractions from each sample) using the primers 8F and 1492R [24] and 21F and 958R,[25] respectively. Amplification of dsrAB gene from community DNA was performed using the degenerate primers DSR1F and DSR4R.[26] Clone library analysis Clone libraries were constructed with amplified 16S rRNA and dsr genes. PCR products (16S rRNA and dsr genes) were purified using gel-purification kit (Qiagen, Venlo, The Netherlands), cloned into pGEM-T easy vector (Promega, Madison, WI, USA) and transformed into E. coli JM109 following manufacturer’s instructions. Randomly selected positive clones were used and assembled for individual clone library. From each library, respective genes were reamplified by colony PCR using vector specific primers SP6 and T7. Colony PCR products were purified and digested with restriction enzymes (HaeIII and RsaI) in separate reactions. Reaction products (10 µL) were run on 2.5% agarose gel for 3 h. Gels were photographed, and band patterns were compared visually. Similar restriction pattern was referred as an Operational Taxonomic Unit (OTU) or ribotype. At least one representative clone from each dominant OTU was selected for nucleotide sequencing. Statistical analyses Shannon diversity index (H) and evenness (EH ) were calculated from clone library data. Simpson’s diversity indices (1/D) were calculated to quantify the diversity of phylotypes.[27] Rarefaction curves were plotted by Analytic Rarefaction 1.3 to assess the diversity coverage based on number and frequency of ribotypes identified in each library. Principle component analysis (PCA) and clustering methods were implemented using MATLAB version 7:8:0. Among the four different metrics (sqEuclidean, cityblock, cosine and correlation) available in MATLAB, cosine matrix was used in this study. A comparison between the bacterial community composition within the three test samples as well as samples from U-contaminated sediments and other mine sites were analyzed using the Pearson’s correlation coefficient method and displayed graphically as a dendrogram based on an unweighted pair group method with arithmetic average (UPGMA) algorithms. Phylogenetic analysis For 16S rRNA gene about first 600–700 nucleotides of representative clone from each dominant OTU were sequenced. Potential chimeras of all the sequences were examined with the CHIMERA CHECK program at the Ribosomal Database Project II using default settings. Both BLAST program of NCBI database and hierarchy browser

from Ribosomal Database Project II were used to find out nearly identical sequences for the 16S rRNA genes. Phylogenetic lineage of all bacterial groups detected within these samples was ascertained by retrieving similar sequences previously reported from uranium ore/mines or heavy metal/radionuclide contaminated sites as well as sequences of type strains retrieved from RDP databases. Phylogenetic trees were constructed using the neighbor-joining method with Jukes-Cantor distance in MEGA4. For the dsr genes, phylogenetic analysis was also done using similar sequence obtained from NCBI databases. Nucleotide sequence accession numbers Nucleotide sequences obtained in the present study were deposited in the GenBank under the following accession numbers: bacterial 16S rRNA genes HQ909283HQ909337, HM469536-HM469553; archaebacterial 16S rRNA gene JF509139-JF509155 and dsr gene JF751011JF751022.

Results Physicochemical and microbiological analysis Physicochemical analyses (Table 1) showed that fresh tailings (JFT244) with their higher pHs contain low phosphorus, sulfur, potassium and nitrogen compared to vegetated tailings (JOT245) and mine sediment (J1-5). The latter samples had relatively higher N, P and K, acidic and neutral pH, respectively. Both tailings samples showed higher conductivity and characteristically low organic carbon. Abundance of heavy metals and actinides (e.g., Ni, Zn, Cu, Cr, U and Th) was detected in all three samples. Relatively higher amounts of several metals including U were found in the fresh tailings. Microbiological counts as enumerated by determining colony forming units grown over various nutrient media showed highest numbers in R2A medium. With respect to cultivable heterotrophic bacteria, mine sediment with its higher organic C, supported highest counts (the order of 109 cells g sample−1) followed by vegetated tailings (108 cell g sample−1) and the least in the fresh tailings (in the order of 105 cell g sample−1). Microbial diversity analysis Microbial diversity within these samples was ascertained by analyzing both bacterial and archaebacterial 16S rRNA gene diversity using community metagenome-derived 16S rRNA gene clone libraries. For samples JFT244, JOT245 and J1-5, total 139, 153, 71 and 65, 58, 77 clones representing bacterial and archaebacterial 16S rRNA genes, respectively, were analyzed by ARDRA (amplified ribosomal DNA restriction analysis). Shannon diversity indices of bacterial clone libraries showed considerably

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higher values (H > 3, EH > 0.9) indicating high diversity and homogeneous community composition, particularly in mine sediments. In contrast, 16S rRNA genes from archaebacterial members showed substantially reduced diversity. Richness and equitability (EH ) values as obtained for both bacterial and archaebacterial libraries indicated that microbial community within the fresh tailings was much less diverse and relatively heterogeneous. Rarefaction analysis (Fig. 1a) indicated distinct saturation for all samples except the one representing the bacterial library for sample J1-5 indicating that both tailings samples were adequately sampled. Microbial community composition Nearly all dominant as well as few minor ribotypes (OTUs) of bacterial and archaebacterial members from six libraries were sequenced to ascertain their taxonomical identity. Phylogenetic affiliation and relative abundance of constituting populations within each community is presented in Fig. 1b. The data showed predominance of Proteobacteria (covering more than 50% of individual community) followed by Acidobacteria (covering 14% to 37% in JOT245 and J1-5 communities) and Bacteroidetes (as a relatively less-abundant group) within the samples. Among the relatively less abundant and frequent populations, Cyanobacteria, Firmicutes, Chloroflexi, Planctomycete, Actinobacteria and Genera incertae sedis OP10 were detected in tailings samples while Genera incertae sedis TM7, Gemmatimonadetes and Unclassified bacteria were found in the mine sediment. The archaebacterial populations revealed abundance of Crenarchaeota within the tailings samples where as members of Euryachaeota, constituted the predominant group in the mine sediment. Phylogenetic analysis (eubacteria) Gammaproteobacteria. Members of γ -Proteobacteria were most abundant in fresh tailings sample (24%) followed by mine sediments (11%). However, in vegetative tailings they were less represented. Members of this subdivision as retrieved from JFT244 were found to be affiliated with families Xanthomonadaceae, Legionellaceae and Pseudomonadaceaec (Fig. 2). Sequences of several OTUs from JFT244 showed their affiliation with genera Frateuria and Dyella or Legionella of Xanthomonadaceae or Legionellaceae, respectively. Family Pseudomonadaceae was represented by bacteria having strong lineage with uncultured Pseudomonadaceae clones previously retrieved from U-ore deposits. Presence of bacteria related to purple sulfur bacterium Ectothiorhodosinus mongolicus and aquatic (hypersaline) Saccharospirillum spp. were found within vegetated tailings (JOT245) and mine sediment (J1-5), respectively.

Fig. 1. (a) Rarefaction curves for bacterial and archaebacterial ribotypes retrieved from uranium mine tailings and mine water sediment. Curves JFT244-B, JOT245-B, J1-5-B are for libraries of bacterial origin while curves JFT244-A, JOT245-A, J1-5-A are for libraries of archaeal origin. (b) Frequency distribution of bacterial and archaebacterial groups (at phylum or class level) within U mine tailing and mine water samples.

Betaproteobacteria. Bacterial groups affiliated with this class were detected in all three samples but with varied abundance [JFT245 (29%) > JOT244 (21%) > J1-5 (12%)]. Members of Betaproteobacteria were found to be represented by families Hydrogenophilaceae, Methylophilaceae, Rhodocyclaceae and Burkholderiales incertae sedis

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Fig. 2. Phylogenetic tree of γ -Proteobacteria related 16S rRNA gene sequences. Clones whose designations include JFT244, JOT245 and J1-5 are analyzed in the present study.

(Fig. 3). A number of sequences from tailings samples showed relatedness with sulfur-, iron- and thiosulfateoxidizing autotrophic Thiobacillus (Hydrogenophilaceae) especially, with T. thioparus, T. denitrificans and few uncultured Thiobacillus previously reported from lead-zinc mine tailings or uranium mill tailings. Presence of bacteria closely related to aerobic methylotrophic bacterium Methylophilus leisingerii and uncultured Methylophilus sp. retrieved previously from ultradeep mines was detected within fresh tailings. Family Rhodocyclaceae, although present in both tailings, was found to be a major group representing 15% of total community in vegetated tailings. Within this family, sequences were related to either perchlorate-reducing bacterium Dechloromonas or with uncultured Rhodocyclales members previously retrieved from iron-oxidation biofilmsU mine waste pile and N2 -fixing Azonexus fungiphilus. Family Burkholderiales incertae sedis detected in JFT244 as well as J1-5 was represented by Methylibium sp., uncultured iron-reducing organisms and Burkholderiales bacterium.

Alphaproteobacteria and deltaproteobacteria. Members of α-Proteobacteria although were detected in all three samples, they represented relatively higher proportions in fresh tailings (18%) and vegetated tailings (12%). Within this class, bacterial groups affiliated to families Rhodospirillaceae, Phyllobacteriaceae, Acetobacteraceae, Methylocystaceae and Hyphomicrobiaceae were detected (Fig. 4a). Family Rhodospirillaceae was represented well by sequences from both fresh as well as vegetated tailings showing affiliation with N2 -fixing Azospirillum sp., particularly with A. lipoferum previously retrieved from U mine tailings and an Azospirillum reported from heavy metal contaminated soil. Members of the Phyllobacteriaceae present in both mine sediment and fresh tailings were affiliated to iron-oxidizing, nitrate-reducing bacterium Parvibaculum lavamentivorans. The presence of bacteria closely related to Acidocella sp. previously retrieved from copper mine was noticed. Families Methylocystaceae and Hyphomicrobiaceae were represented by two and three ribotype from vegetative tailings, respectively. Class δ-Proteobacteria was found in

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Fig. 3. Phylogenetic tree of β-Proteobacteria-related 16S rRNA gene sequences. Clones whose designations include JFT244, JOT245 and J1-5 are analyzed in the present study.

mine sediment (J1-5) and vegetative tailings (JOT245). Sequences under this clade showed lineage with sequence of uncultured Desulfuromonadales bacteria and Geobacter spp. retrieved earlier from iron-reducing anoxic and mineimpacted lake sediments (Fig. 4b). Acidobacteria. Members of this phylum were found to be one of the most abundant groups in mine sediment and vegetative tailings representing nearly 21% and 10% of respective communities. They were also detected in fresh

tailings but with much less (2%) abundance. Sequences affiliated to Acidobacteria were found to be distributed in three subgroups, namely Gp1, Gp4 and Gp9. As evident from the Neighbor-Joining tree (Fig. 5), two ribotypes from JFT244 and JOT245 were within subgroup 1 (Gp1), with sequences showing lineage to uncultured Acidobacteria previously retrieved from coal mine or copper mine drainage or from hydrocarbon contaminated soil. Subgroup 4 (Gp4) was solely detected from the mine sediment with sequences (representing the most abundant as well as

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Fig. 4. Phylogenetic tree of α (a)- and δ (b)-Proteobacteria related 16S rRNA gene sequences. Clones whose designations include JU and BJ were analyzed in the present study.

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Fig. 5. Phylogenetic tree of Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes and other lessabundant bacteria related 16S rRNA gene sequences. Clones whose designations include JFT244, JOT245 and J1-5 are analyzed in the present study.

several other ribotypes) showing their lineage with uncultured Acidobacterium previously retrieved from polyaromatic hydrocarbon contaminated soil. A single ribotype, JOT245-142 from vegetated tailings of subgroup 9 (Gp9) showing its closeness with uncultured Acidobacteria previously retrieved from uranium contaminated subsurface soil. Less abundant groups. Members of bacterial phyla Bacteroidetes, Actinobacteria, Firmicutes, Chlorophyta, Cyanobacteria, Chloroflexi, Planctomycete and Gemmati-

monadetes were relatively less abundant across the communities. Members of Bacteroidetes detected in all three samples but represented the most abundant phyla within the fresh tailings. As evident from the phylogenetic tree (Fig. 5), this phylum was represented by members of families Chitinophagaceae and Cytophagaceae. Sequence of the major ribotype JFT244-129 from the fresh tailings showed its strong lineage with Sediminibacterium sp. and also with several other uncultured Bacteroidetes previously reported from heavy metal-contaminated samples.

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Microbial communities in uranium mine tailings Two sequences retrieved from vegetative tailings were found to be affiliated to this family showing relatedness with uncultured Niastella sp. and uncultured Bacteroidetes retrieved previously from uranium mining wastes. Sequence of a minor ribotype of mine sediment showed its strong lineage with uncultured Bacteroidetes bacterium from uranium mining wastes. Members of Actinobacteria and Firmicutes were detected either in fresh tailings or vegetated tailings, respectively. Phylum Firmicutes was found to be one of the most abundant groups in the later sample represented by families Bacillaceae and Clostridiaceae. Sequences affiliated to these two families showed strong lineage with genera Bacillus and Clostridium as well as with uncultured bacteria/spp. previously obtained from various U mining wastes. Member of the phylum Cyanobacteria was detected only in fresh tailings with ribotype JFT244104 showing its lineage to uncultured Cyanobacterium previously retrieved from Bagjata uranium mine (Fig. 5). Members of Chloroflexi and Planctomycete were detected only in vegetated tailings with sequences having close relatedness with Chlorobi and uncultured Planctomycete previously retrieved from uranium mine waste pile/mill tailings, etc. Members of the phylum Gemmatimonadetes was detected only in mine sediment showing lineage with uncultured Gemmatimonas sp retrieved from Anderson Lake (Santa Clara County, California, USA). Phylogenetic analysis (archaebacteria) Members of the domain Archaea were detected within all the three samples and were found to be represented by phyla Crenarchaeota and Euryachaeota. As evident from phylogenetic analysis (Fig. 6), sequences representing ribotypes under Crenarchaeota were all affiliated to class Thermoprotei and remained segregated into three clusters. Cluster one and two were composed of sequences from the tailings showing close lineage with sequence(s) of uncultured Crenarchaeota previously retrieved from uranium mill tailings, volcano mud/aspen rhizosphere and uncultured archaea from hydrocarbon contaminated soil, respectively. The third cluster was composed of sequences representing five ribotypes from vegetated tailings covering 34% of archaeal community and showing their strong lineage with uncultured Crenarchaeota retrieved earlier from sewage wastewater and from oyster shell. Phylum Euryachaeota was represented by sequences of several ribotypes from mine sediment and fresh tailings. All the affiliated sequences under this phlum were members of class Methanomicrobia. Under this class, sequence of the ribotype JFT24468 showed strong lineage with (100% bootstrap value) Methanosarcina lacustera and other species retrieved earlier from anoxic lake sediments and sedimentary rock. Two ribotypes from mine sediment (including the most dominant one) showed their close lineage with Methanobacterium thermoautotrophicum: a heavy-metal-tolerant methanogen

isolated previously from a waste-disposal site and uncultured Methanosarcinales previously retrieved from a biodegraded, high-temperature petroleum reservoir, respectively. Analysis of dissimilatory sulfite reductase gene The dissimilatory sulfite reductase (dsr) gene of sulfatereducing bacteria (SRB) play an important role in metal including uranium geochemistry and have been found to be a useful tool for bioremediation of metals and radionuclides from contaminated environments. To assess the composition of sulfidogenic populations within the studied samples, diversity of dissimilatory sulfate reductase gene (dsrAB) was analyzed. Among the samples, only vegetative tailings (JOT245) yielded positive amplification of the target gene. Restriction pattern analysis of cloned dsr gene from this sample showed presence of 12 RFLP groups within the library of 70 clones. Sequence analysis confirmed their affiliation with either dsrA or dsrB genes. Among the five dsr gene sequences of most abundant and few other RFLP groups (together covering more than 50% of the library), strong similarity with dsrA gene obtained previously from lake sediment or from paddy soil was noticeable. Phylogenetic analysis, however, indicated their close lineage with similar sequences from SRB (sulfate-reducing bacteria) previously retrieved from both uranium-mining sites and paddy fields forming a clade with 100% bootstraps support (Fig. 7). Another sequence representing a less-abundant RFLP group showed close lineage with Desulfotomaculum aeronauticum dsrA gene retrieved earlier from uranium mill tailings of Shiprock, USA. Sequences representing two distinct ribotypes were closely related to dsrB sequence of either uncultured SRB from paddy soil or with D. putei and D. nigrificans previously retrieved from uranium mill tailings. Four sequences representing the RFLP groups that together covered 23% of the library although showed similarity with dsrB sequences obtained previously from freshwater vegetated intertidal soil or paddy soil, phylogenetic study indicated their close lineage with dsr from uncultured Syntrophobacter sp. retrieved earlier from denitrifying sulfide removal granule. The remaining five dsrB sequences branched out and formed a single clade showing a strong lineage with uncultured Syntrophobacter sp. from denitrifying sulfide removal granule. Statistical analysis PCA and clustering was used to study the correlation among the samples, if any, with respect to microbial community composition and their physicochemical nature. To ascertain the possible impact of U mine waste contamination, data of a few additional samples collected from nearby mine sites, but devoid of any contamination, were also included. Biplots made by PCA followed by

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Fig. 6. Phylogenetic tree of Archaea related 16S rRNA gene sequences. Clones whose designations include JFT244, JOT245 and J1-5 are analyzed in the present study.

Fig. 7. Phylogenetic tree of dsr gene sequences. Sequences retrieved during this study are marked as DSR245.

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Fig. 8. Two-dimensional plots of principal components analysis of the total measured physicochemical (a) and bacterial groups (b) as detected by 16S rRNA gene sequences across the contaminated tailings pond and mine sediments with relatively non contaminated agricultural soil samples.

cluster analysis using the physicochemical and bacterial diversity data revealed the presence of three similar and distinct clusters (Fig. 8). Samples from non-contaminated sites formed a separate cluster showing their marked difference from vegetated tailings and mine water. The highly contaminated,

low-nutrient fresh tailings in such analysis remained distinctly away from both these groups. The strong positive correlation as obtained from this PCA analysis indicated that physicochemical environment within the three study sites had significant impact in shaping the microbial community.

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Fig. 9. UPGMA cluster analysis of bacterial groups detected in the present samples as well as samples studied earlier from a similar environment.

UPGMA-based analysis of bacterial community composition within the samples obtained from different other mine tailings and uranium/metal contaminated sites was performed to compare the diversity patterns (Fig. 9). As evident from the figure, the UPGMA plot showed a close agreement between the three test samples from Jaduguda, bacterial diversity reported earlier from U ore of Jaduguda and the uranium waste contaminated site at Oak Ridge (Tennessee). Interestingly bacterial diversity from Pb-Zn mine tailings and two other U-contaminated sites remained distinctly separated from the former group.

Discussion The culture-independent survey of microbial community in highly hazardous U mine tailings and mine sediment provides critical insights in geomicrobiology of these sites essential for their long-term remediation. It was observed that, despite considerably high level of contamination, these samples harbor microbial abundance and broad phylogenetic diversity. The fresh tailings with characteristic radioactivity, higher pH, low organic carbon and P, S, N as well as elevated concentration of heavy metals including radionuclides represented an extreme and oligotrophic environment. In spite of their high metal content, vegetated tailings and mine sediment on the other hand showed relatively

higher concentrations of nutrients. It is interesting to note that physicochemical conditions as prevailed in the test samples had observable, significant impact on microbial abundance, diversity and community composition. The fresh tailings with their extreme nature allowed lowest microbial abundance and diversity (as detected by CFU counts and Shannon diversity indices, respectively), while the other two samples having higher organic carbon and nutrients were possibly selected as better habitats for diverse microbes. Organic carbon, pH and moisture content are known to have strong influence on inhabitant microorganisms by affecting bioavailability of metal ions and/or by enhancing microbial metabolic potential to withstand metal toxicity.[28] Increase in microbial diversity along with high TOC has been previously detected in several mine environments.[14] Although no attempt was made to measure total microbial biomass, but based on the culturable heterotrophic bacterial population sizes, it can be concluded that the presence of higher organic carbon and nutrients (e.g., N, P and S) favored the microbial communities to flourish within the sites, overcoming the toxicity imposed by higher metal and radionuclide content. It was evident that bacterial community in fresh tailings was mainly composed of organisms having strong lineages with chemoorganotrophic genera Frateuria, Legionella, and chemoautotrophic Thiobacillus along

Microbial communities in uranium mine tailings facultative methylotrophic Methylophilus, well known for their metal and sulfur oxidizing and/or metal resistance ability. Most of these bacterial groups have previously been reported from U ore as well as U, Au, Cu, Pb and Zn mine tailings and have shown potential role in metal/radionuclide biogeochemistry.[9,17,29] Abundance of several bacterial groups within the fresh tailings capable of using inorganic electron donors or assimilate N2 seems in line with the chemical nature of this sample; i.e., lack of sufficient but availability of sulfur and heavy metals those might act well as electron donor or acceptor. Significant presence of aerobic nitrogen-fixing, phosphate solubilizing Azospirillum and uranium sequestering group Burkholderiales incertae sedis within this sample is noticeable. The genus Azospirillum is not only known for its plant growth-promoting role by supplying essential N and P nutrients to the soil but also found to have important function in radionuclide bioremediation.[30] Members of Burkholderiales incertae sedis as well as several nonproteobacterial genera found in fresh tailings (like Sediminibacterium, Cellulomonas, Chlorella and Cyanobacteria) as detected here have also been retrieved earlier from U contaminates sites and are known for their U removal capacity.[9,10,31] Together with diverse chemolithotrophic and N2 -fixing bacterial populations, anaerobic -methanogenic Methanosarcina and -chemolithotrophic Thermoprotei possibly represents characteristic synotropic assemblage within the tailings capable of providing critical supply of nutrients within the ecosystem for other members of this community. Interestingly, the microbial community within the vegetated tailings was considerably more diverse with presence and abundance of heterotrophic as well as few chemo-lithoautotropic bacteria including purple sulfur bacteria, N2 -fixing genera and nitrate, sulfur, iron and metal-reducing groups. Presence of Gram-positive metalresistant and -sequestrating genera Clostridium, Bacillus as well as metal/nitrate/sulfur-reducing anaerobic groups (like Dechloromonas, Azonexus, Hyphomicrobium, Desulfuromonadales and Geobacter) very well known for bioprecipitation of U and other metal/radionuclides is highly significant. Distinct abundance of Acidobacteria in this sample could be inferred as a result of altered physicochemical properties of this sample; in particular, pH and organic carbon. Higher abundance of this group of uncultured bacteria in relatively -less contaminated and -nutrient rich vegetated tailings seems in line with previous reports that showed increase abundance of this phylum with rise in organic carbon content and decrease in pH towards neutrality.[28,32] Abundance of all these organisms possibly indicates the community’s ability for precipitation and thus immobilization of soluble metals/radionuclides. The later fact remains more apparent with detection of Chloroflexi, well known for establishing syntrophic relationship with sulfate-reducing bacteria during active bioremediation.[31] The role of sulfate reduction process within the vegetated tailings gets further substantiated due to the successful detection of dissimila-

707 tory sulfate reductase (dsr) gene from its metagenome. Particularly the affiliation of dsr genes to δ-Proteobacteria, including Desulfotomaculum sp. and syntrophic propionatedegrading sulfate-reducing Syntrophobacter sp. has been documented is most notable. Abundance of Desulfotomaculum sp. is reported earlier from U mill tailings disposal site, whereas members of the genus Syntrophobacter is known to have the capacity to replace the sulfidogenic metabolism with a syntrophic metabolism that depends on the presence of a methanogenic organism and therefore could play a significant role in the biogeochemical cycle.[33] The presence of methanogenic organisms also has been reported in this study, and therefore existence of a symbiotic community within the vegetated tailings is possible. The data indicated that a microbial sulfate reduction process might be taking place within these samples, thereby suggesting that the major radionuclide contaminants may potentially be immobilized in situ with such microbial activity. In comparison to the tailings samples, mine sediment showed higher bacterial counts and diversity. Bacterial community in this sample is dominated by Acidobacteria, Methylibium, Gemmatimonas, Parvibaculum, Saccharospirillumm, Desulfuromonadales etc. In spite of high metal/radionuclide’s content, increased abundance of Acidobacteria as one of the most predominant members of natural ecosystems including those contaminated with metals is highly conspicuous. Although these organisms are well known for tolerating extreme conditions, including metal contamination and acidic pH [34] their ecological role remains unknown.[10] However, the ubiquity and abundance of Acidobacteria as well as their ability to withstand polluted and extreme environments suggest that they serve functions important in the environment and are potentially quite varied.[35] Our result indicated that tailings pond samples with lower pH and lower organic carbon content harbor Acidobacteria subgroup 1, while members of subgroup 4 is present in the mine sediment, which showed relatively higher pH and level of organic carbon. The later finding corroborates well with previous results wherein sequences affiliated to Acidobacteria subgroup 4 have been found to be abundant in soils of neutral or alkaline pH (in 12 different soils) but their presence was variable in soils with pHs below 5.4, and they were not detected in soils with pHs of less than 4.0.[35] Members of bacterial genera Parvibaculum and Desulfuromonadales bacteria, as also found in vegetated tailings, are well-known heterotrophic acidophilic organisms capable of sulfate, nitrate, and Fe(III)-reduction using a variety of electron donors such as acetate, alcohols and aromatic compounds.[10,36,37] The ecological function of Methylibium and Saccharospirillum present in this sample remains yet to be established. Presence of anaerobic methanogenic Methanosarcina and chemolithotrophic Thermoprotei of the archeal domain in this sample is notable.

708 Conclusion The study reports microbial community structure and composition within highly contaminated U mine tailings and mine water sediment from the active uranium mine. Cultivable bacterial counts (CFU), as well as diversity indices, and abundance of specific bacterial groups as detected through a culture-independent approach showed a distinct effect of the sample’s physicochemical nature on microbial abundance and diversity. In particular, with increasing metal and reducing nutrient (organic C, N, P, etc.) contents, microbial counts and diversity decreased significantly. Principal component analysis further revealed a strong correlation among the geochemical properties of the samples and bacterial groups present therein. Our findings revealed that while the tailings samples were dominated by chemolithotrophic, N2 fixing, S and metal oxidizing, as well as methylotrophic genera, in contrast, the organic C-rich mine sediment harbor more of Acidobacteria along with metal resistant, and metal reducing populations. Noticeably, the presence of specific bacterial groups involved in Fe, S and CH4 geocycling were more prevalent in fresh tailings, whereas the vegetated tailings harbor organisms catalyzing dissimilative metal and SO4 reduction cycle, dissimilative anoxic sulfur cycle and methane cycling. The study provides the first culture-independent analysis of autochthonous bacterial and archeal populations within the mine tailings and mine sediment, providing critical insight on biogeochemical activities relevant for long-term stabilization of such hazardous environments.

Funding The authors gratefully acknowledge the financial support from Board of Research in Nuclear Sciences, Department of Atomic Energy, Government of India. Kind support during the field work from Uranium Corporation India Ltd., Jaduguda, is thankfully acknowledged.

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