Current Microbiology (2019) 76:462–469 https://doi.org/10.1007/s00284-019-01640-9

Insights into the Bacterial Diversity of the Acidic Akhtala Mine Tailing in Armenia Using Molecular Approaches

Armine Margaryan1,2,3 · Hovik Panosyan1 · Chonticha Mamimin3,4 · Armen Trchounian1,2 · Nils‑Kåre Birkeland3

Received: 24 July 2018 / Accepted: 24 January 2019 / Published online: 18 February 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract The impact of the heavy metal contamination and acidity on the bacterial community was studied in samples collected from the Akhtala copper mine tailing using molecular approaches. The bacterial community structure analysis by PCR-DGGE fingerprinting revealed an abundance of Firmicutes, Acidobacteria, and Proteobacteria in different layers of the Akhtala tailing. 454 pyrotag sequence analyses revealed that a significant part of the sequences originated from Proteobacteria (49%) and Bacteroidetes (43%). Bacterial taxa are distributed also in phyla Saccharibacteria (2%), Verrucomicrobia (1.5%), Gam- matimonadetes (1%), and some minor additional bacterial groups. The main primary producers in the Akhtala tailing appear to be obligate autotrophic Thiobacillus and Sulfuritalea species. Representatives of Lutibacter and Lysobacter genera are the most abundant acid-tolerant heterotrophs in the studied tailing. The presence of a large number of yet-uncultivated species emphasizes the importance of the future exploration of the tailing as an important source of novel bacteria.

Introduction the soil ecological balance, including the microbial diversity, and induce further disruption of the ecosystem [1, 7, 27, 42]. The soil environment is a massive pool for a variety of The environmental stress caused by heavy metals generally chemicals and heavy metals, which eventually leads to produce a change of the native bacterial community leading environmental contamination problems. Indeed, different to a numerously domination of metal-tolerant microorgan- types of heavy metals are used and emitted in huge amounts isms [23, 25, 38]. A list of bacterial groups has adapted through various androgenic activities including mining, and developed abilities to deal with toxic levels of metals smelting, and metal forging [27]. Millions of tons of trace through detoxification mechanisms, metal absorption, peri- elements are produced every year from mines in demand for plasmic or intracellular accumulation, extracellular precipi- new materials [46]. Through emanation into the environ- tation, and efflux of heavy metals from the cells29 [ ]. The ment, the heavy metals get accumulated and may damage phenomenon of microbial heavy metal resistance has major importance and is crucial in microbial ecology, especially in processes of biogeochemical cycling of heavy metals and Electronic supplementary material The online version of this article (https​://doi.org/10.1007/s0028​4-019-01640​-9) contains in the bioremediation of metal-contaminated ecological sys- supplementary material, which is available to authorized users. tems [27, 35, 43]. The study of microbial diversity in different environments * Nils‑Kåre Birkeland based on culture-based approaches can provide useful infor- [email protected] mation on the diversity of microbial communities [6, 48]. 1 Department of Biochemistry, Microbiology However, various environmental studies have demonstrated and Biotechnology, Yerevan State University, 0025 Yerevan, that < 1% of the total microbial community members are Armenia cultivable under laboratory conditions and applying culture- 2 Research Institute of Biology, Yerevan State University, dependent methods will thus only reveal information about 0025 Yerevan, Armenia a minor fraction of the microorganism [17, 39]. Molecular 3 Department of Biological Sciences, University of Bergen, approaches such as genetic fingerprinting, metagenomics, 5020 Bergen, Norway metaproteomics, and metatranscriptomics are now consid- 4 Department of Industrial Biotechnology, Faculty ered vital for understanding of the vast diversity of microbes of Agro‑Industry, Prince of Songkla University, Songkhla 90112, Thailand

Vol:.(1234567890)1 3 Insights into the Bacterial Diversity of the Acidic Akhtala Mine Tailing in Armenia Using… 463 in nature and for understanding their interactions with biotic 41°09.356′ E 44°45.948′) (Fig. 1). The pH and conductivity and abiotic environmental factors [22, 23, 33, 34, 48]. were measured using a portable potentiometer (HANNA, Bowels of Armenia have significant reserves of copper, HI98129 pH/Conductivity/TDS Tester) after the sludge sam- molybdenum, and gold, as well as lead, silver, and zinc. Cur- ples had been suspended in distilled water (w/v, 1: 2.5). The rently, Armenia harbors about 107 mineral ores out of which mineralization and pH, as well as concentrations of sulfate, 26 are still being exploited. Development of mining and chloride, and heavy metal ions in the mine tailing outlet smelting industries leads to increasing emissions of large to the river flowing next to the tailing, were measured by amounts of waste into the environment, especially through an Elan 9000 ICP mass-spectrometer with indium internal formation of heavy metal-containing mine tailings, which standard and argon plasma (Table 1) [8]. Collected samples are primary sources for heavy metal pollution in Armenia were stored in a cooling bag and transported to the labora- [13, 37]. Iron-oxidizing chemolithotrophic bacteria belong- tory for DNA extraction. ing to Acidithiobacillus, Leptospirillum, and Sulfobacillus genera, as well as metal-resistant bacilli belonging to Bacil- DNA extraction and Denaturing Gradient Gel lus, Brevibacillus, and Geobacillus genera were previously Electrophoresis (DGGE) isolated from the complex ore samples of the Akhtala, Sotk, Kajaran, and Kapan mines [18, 41]. However, there is lack of DNA was extracted from 0.5 g sludge samples taken from understanding of the abundance of different bacterial popu- the surface and 10-cm-deep layers, as well as from mixed lations in the Armenian mines. The objective of this study samples of the tailing. The DNA extraction was performed was to determine the bacterial community structure in the as previously described [50]. Analyses of extracts by elec- sludge samples from the Akhtala tailing based on molecular trophoresis on a 0.7% agarose gel containing 0.01% GelRed approaches. was used to estimate DNA quantity and quality. 16S rRNA gene PCR-DGGE was performed as described by [28]. The extracted DNA was used as templates for ampli- Materials and Methods fication of the V3 region of 16S rRNA gene using primer L340F (CCT​ACG​GGA​GGC​AGCAG) with a GC clamp Sampling and Chemical Analyses (GCG​CCC​GCC​GCG​CGC​GGC​GGG​CGG​GGC​GGG​GGC​ ACG​GGGGG) and K517R (ATT​ACC​GCG​GCT​GCTGG) Samples were taken from the surface and from 10-cm- [30]. The PCR reaction mixtures of a final volume of 50 µl deep layers of the Akhtala copper mine tailing located in contained ≈ 100 ng DNA, 10 µl 5 ×OneTaq standard reac- Armenia’s Northern Lori province, near Akhtala town (N tion buffer, 1 µl 10 mM dNTPs, 0.5 µM of each primer, and

Fig. 1 The location of Akhtala tailing in the map (a) and tailing overview (b). The red pointer shows the location of the tailing on the map; arrow shows the sampling site

Table 1 The chemical properties of the water samples collected from the river contaminated with the sludge of the Akhtala tailing Mineralization (µS/cm2) Sulfate ions (mg/l) Chloride ions (mg/l) pH Heavy metal concentrations (mg/l) Cr Fe Mn Co Ni Cu Zn Cd

1437 672.74 20.83 2.5 0.0035 6.55 0.98 0.01 0.0082 0.498 5.87 0.04

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0.25 µl (1.25 units/50 µl) OneTaq DNA Polymerase (Bio- estimated. A taxonomic assignment of the reads was gener- Labs, New England). The reaction profile was the following: ated using the Silva SSU database. initial denaturation at 94 °C for 3 min followed by 30 cycles of three steps (94 °C for 1 min, 55 °C for 30 s, and 68 °C for Nucleotide Sequence Accession Numbers 1 min) and a final extension at 68 °C for 10 min. PCR products were analyzed using the TV-400-DGGE These 16S rDNA Pyrosequencing data have been depos- System (Topac Inc., USA) with 8% (w/v) polyacrylamide ited at NCBI GenBank under the accession number gel (37.5:1 acrylamide/bisacrylamide) in 0.5x TAE (20 mM SAMN06062036. Tris–HCl, 10 mM Acetat, 0.5 mM EDTA) buffer and a dena- turant gradient of 30–70% (100% denaturant contained 7M urea and 40% deionized formamide). Electrophoresis was Results performed at a constant voltage of 20 V for 10 min, followed by 200 V for 4 h. DGGE gels were stained with SYBR®Gold Both samples from the Akhtala mine tailing were highly (Invitrogen, USA) for 60 min and photographed on a Gel acidic (pH 2.6) with a relatively high dissolved mineral DocXR system (Bio-Rad Laboratories). Bands were excised content (7700 µS/cm). Denaturing gradient gel electropho- from the gel and gel slices were incubated in 20 ml of Mil- resis (DGGE)-based analysis of the extracted environmen- liQ water at 4 °C for 24 h to promote DNA elution, and then tal DNA was done to provide a snapshot of the dominating re-amplified by PCR as described above with the mentioned microbial populations. DGGE patterns showed the occur- primer set without GC clamp. Re-amplified PCR products rence of complex bacterial communities in the analyzed were purified with GenElute™ PCR Clean-up Kit (Sigma) sample (Fig. 1S). To identify the dominant taxa associated and sequenced. with the PCR-DGGE profiles, DGGE bands were extracted from the gel and sequenced after re-amplification of the 16S Sequencing and Phylogenetic Analysis rRNA gene fragment. Successful sequencing of the partial 16S rRNA gene could only be obtained for 16 of the bands. Most of the bacteria detected by DGGE fingerprinting of Sanger sequencing of re-amplified PCR products was per- the Akhtala tailing belonged to the phylum Proteobacteria formed using the BigDye Terminator kit (Perkin Elmer) and (Table 1S) and shared less than 97% identity with their clos- an ABI PRISM capillary sequencer. The absence of chi- est match in GenBank, indicating a unique community with meric sequences was verified using the DECIPHER web tool many potential novel species. (http://decipher.cee.wisc.edu/FindC​ himer​ as.html​ ) [45]. Raw In the surface layer of the Akhtala tailing (bands 1–6), sequence data were handled with the program Chromas Lite the prevailing bacteria belonged to the phylum Firmicutes, 2.1.1 (by Australia Technelysium Pty Ltd) and BioEdit [16]. clustering in the genus Bacillus. In the 10-cm-deep layer The closest matches for partial 16S rDNA sequences were (bands 7–10), the dominating bacteria belongs to the phylum identified by the basic local alignment search tool (BLAST) Acidobacteria. The partial 16S DNA sequences derived from against the National Center for Biotechnology Information band 11 and 13–17 are closely related to the class Gam- database (NCBI; http://blast​.ncbi.nlm.nih.gov/Blast​.cgi). maproteobacteria, genus Pseudomonas. This indicates a unique structure and special stratification of the bacterial Pyrosequencing and Pyrotag Analysis community in the studied sample. In order to provide a more detailed analysis of the bacte- The V4–V8 variable regions of the bacterial small subunit rial community in the Akhtala tailing, total DNA extracted 16S rRNA gene were PCR amplified with the ‘‘universal’’ from a mix of the sludge samples was analyzed by tagged primer set (8-28F: TGATCC​ TGG​ CTC​ AG)​ and K517R using 454 pyrosequencing. After quality control and trimming, a two-step PCR protocol [5]. In the second PCR, a special about 12,000 high-quality reeds were recovered, compris- primer set with m/tag and tag adaptor was used: TitA8-28fF ing 262 OTUs, with Shannon diversity entropy of 5.44. and TitB-K517R. Resulting amplicons were cleaned using This indicates a significantly higher microbial richness and AMPure XP (Beckman Coulter, Oslo, Norway) following diversity than suggested by the DGGE analysis. A total of the manufacturer’s protocol. Pyrosequencing was performed 15 bacterial phyla were identified (Fig. 2). A major part of at the Norwegian High-Throughput Sequencing Centre in the sequences originated from Proteobacteria (49%) and Oslo using Lib-L chemistry and GS-FLX Titanium technol- Bacteroidetes (43%), which are responsible for biomass ogy (454 Life Science, Roche, Penzberg, Germany). degradation and fermentation. Minor bacterial taxa were The obtained sequence data were processed by CLC distributed in phyla Saccharibacteria (2%) (formerly known workbench 8.0.1. The number of operational taxonomic as candidate division TM7), Verrucomicrobia (1.5%), and units (OTUs) and the Shannon diversity index (H′) were Gammatimonadetes (1%). The presence of additional phyla,

1 3 Insights into the Bacterial Diversity of the Acidic Akhtala Mine Tailing in Armenia Using… 465

Fig. 2 Relative abundances of phylogenetic groups in the sludge sample derived from the Akhtala tailing. The dominant phylum, class, family, and genus are shown in the figure such as Actinobacteria, Acidobacteria, Cyanobacteria, Fir- diversity indices (Shannon entropy). The low community micutes, and Planctomycetes, accounts for < 1% based on richness (OTUs = 262) and low diversity indices (Shannon single-read assignments (Fig. 2). Interestingly, that dominat- entropy = 5.44) are probably connected with the acidity of ing phylum Acidobacteria in DGGE band present only 1% of the tailing. Teng and coauthors [38] have summarized the pyrotags. In pyrosequencing, the mix of total DNA extracted latest studies of acidic mine drainage environments to pro- from the Akhtala tailing’s samples of the both layers has vide a better understanding of the microbial biodiversity been used. Thus, these layers were not differentiated in the and community assembly in such sites, and concluded that pyrosequencing results. microbial diversity gradually decreases acidification of mine The most abundant Proteobacteria and Bacteroidetes were tailings [22, 38]. assigned to 11 and 1 genera, respectively, which includes PCR–DGGE-based bacterial community analyses classified, as well as non-classified bacterial groups. The revealed the presence of Firmicutes, Acidobacteria, and most abundant genera are presented in Fig. 2. At the class Proteobacteria in the Akhtala tailing. The BLAST analysis level, the prevalent reads are distributed across Flavobacte- of the partial 16S DNA sequences derived from the DGGE riia (34%), Betaproteobacteria (31%), Gammaproteobac- bands revealed the phylogenetic bacterial group Firmicutes teria (19%), and Sphingobacteriia (6%), which belong to in the Akhtala tailing surface layer. The Firmicutes was phyla Proteobacteria and Bacteroidetes (Fig. 2). absent in the deep layer, where Acidobacteria and Proteo- bacteria dominated. Presumably, the members of the phylum Firmicutes in the surface layer of the tailing can originate Discussion from bacterial spores brought in from the surrounding soils by the wind. A consortium of relatives to novel and unculti- To gain insight into the bacterial community structure of the vated bacterial groups constitute the main bacterial popula- Akhtala tailing, both DGGE fingerprinting and 454-based tion of the Akhtala tailing. tagged pyrosequencing approaches were used. This is the The data derived from DGGE fingerprinting are con- first analysis of microbial populations in environments in firmed by the tagged pyrosequencing analysis of the bacte- the territory of Armenia polluted with toxic compound and rial community in the tailing. The latter revealed the abun- explores the potential of microbial resources for their pos- dance of Proteobacteria (49%), Bacteroidetes (43%), and sible exploitation in relevant bioprocesses (bioremediation, some minor phyla (Fig. 2). biosorption, biomining ant, etc.). Proteobacteria are abundant in many ecosystems, as this The microbial diversity in the Akhtala acid tail- bacterial group includes oligotrophic bacteria adapted to ing (pH 2.6) was characterized by both the community live under different environmental conditions [42]. These richness indices (observed OTUs) and the community bacteria have previously been found in several acid mine

1 3 466 A. Margaryan et al. tailings with high metal content (Table 2), which indicates chemically extreme conditions. For example, the Acidobac- a high degree of metal and acid resistance for certain mem- teria are a natural component of acid mine drainage (AMD) bers of the Proteobacteria phylum. The other dominating communities [3, 19, 42], co-dominant in tailing waste from phylum, Bacteroidetes, has also been identified in AMD mining operations such as for uranium [4], lead–zinc [48], environments, e.g., copper mine tailings in Arizona [32], in and copper [9]. The increased availability of genomic and Jinkouling tailing pond, China (pH 2.65) [47], and an acid physiological information, coupled to distribution data in mine drainage at Svalbard [12] (Table 2). The abundance field surveys and experiments, should direct future progress of Bacteroidetes representatives in several acidic metal-rich in unraveling the ecology of this important but still enig- environments indicates that this group is easily adapted to matic phylum [18]. The presence of the members of yet- these extreme conditions and corroborates our results. The uncultured groups of Acidobacteria in the Akhtala tailing acidic pH of the Akhtala tailing and a high dissolved min- emphasizes the importance of the future exploration of this eral content (7700 µS/cm) have also been drivers for devel- mine tailing as an important source of the new and physi- opment of additional acidophilic/acidotolerant and heavy ologically diverse bacteria. metal-resistant bacterial populations, such as members of It has been reported that a number of acidophilic or acid- the Acidobacteria, although they constitute a minor frac- tolerant bacteria are associated with acid generation from tion of the community. The representatives of the phylum sulfide minerals at pH levels below 4 [2, 24]. A large num- Acidobacteria constitute a group of ubiquitous and diverse ber of dominant genera found in the pyrotag reads could bacteria, which were first described as a novel division in be classified as bacteria associated with microbial Fe and 1997 [25]. This phylum is currently recognized as one of S cycling, indicating dynamic Fe and S cycling in the tail- the most widespread and abundant bacterial lineage on the ing. The members of the genus Thiobacillus and Sulfuri- planet. Members of this phylum have been found in environ- talea from the class Betaproteobacteria are the main groups ments with high levels of metals, acidification, and other involved in Fe and S cycling of Akhtala tailing. Most of

Table 2 Comparison of the bacterial diversity in the Akhtala and other acidic mine tailings Source pH Phylum (%) Analysis method References

Akhtala copper mine tailing, Akhtala, Armenia 2.6 Proteobacteria, 49 Pyrosequencing (GS FLX) This study Bacteroidetes, 43 Saccharibacteria, 2 Verrucomicrobia, 1.5 Gammatimonadetes, 1 Mine tailings of Malanjkhand copper project, Bala- < 2 Proteobacteria, 48 Sequence of V4 region of 16S rRNA gene [15] ghat, India Actinobacteria, 18 Chloroflexi, 10 Firmicutes, 6 Proctor Gulch, Casino Mine watershed, Yukon, 3.75 Proteobacteria, NA Illumina MiSeq [40] Canada Cyanobacteria, NA Tailing dump of the Komsomolskaya gold mine in the 2.29 Proteobacteria, NA Shotgun library (GS FLX) [26] Kemerovo region, Russia Actinobacteria, NA Nitrospirae, NA Tailing dam of multi-metal sulfide Dabaoshan mine, 2.4 Firmicutes, 40.59 Shotgun library, paired-end sequencing [49] Shaoguan, Guangdong Province, China Proteobacteria, 21.67 (Illumina Hiseq-2500) Actinobacteria, 19.37 Acid mine drainage at Bjørndalen, Longyearbyen in 2.8–3.5 Proteobacteria, 41 454 Sequencing (GS FLX) [12] Svalbard, Norway Saccharibacteria, 23 Actinobacteria, 8% Bacteroidetes, 8 Acidobacteria, 7 Verrucomicrobia, 6 Chloroflexi, 4 Planctomycetes, 2 Nitrospirae, 1 Copper mine (Mission Mine) site located at Sahuarita, NA Bacteroidetes, 26 Clone library (TOPO TA Cloning® kit) [32] Arizona Proteobacteria, 23 Actinobacteria, 23 Firmicutes, 16 Chloroflexi, 3

1 3 Insights into the Bacterial Diversity of the Acidic Akhtala Mine Tailing in Armenia Using… 467 these microorganisms are mesophilic or thermophilic, fac- Acknowledgements The work was supported by grants CPEA- ultative anaerobic chemolithotrophs, which could oxidize 2011/10081 and CPEA-LT-2016/10095 from the Eurasia Programme of the Norwegian Agency for International Cooperation and Quality ferrous and sulfur compounds and exhibit high tolerance to Enhancement in Higher Education (Diku) to HP and NKB, and ANSEF various metal ions [20, 21, 24]. Research (#Microbio-3869, 4619) to AM. Thiobacillus and Sulfuritalea species are obligate auto- trophs; they cannot grow with organic carbon as an electron Compliance with Ethical Standards and carbon source and satisfy its carbon requirement from Conflict of interest the fixation ofCO ­ 2. Thiobacillus and Sulfuritalea species are The authors declare that they have no conflict of thus most likely the main primary producers in the Akhtala interest. tailing. Thiobacilli spp. also plays an important role in the nitro- gen cycle, due to their ability to combine inorganic sulfur References oxidation with denitrification. Both­CO 2 fixation and deni- trification processes consume hydrogen ions and contribute 1. Almeida WI, Vieira RP, Cardoso AM, Silveira CB, Costa RG, to making the environment less acidic [14, 24]. Also the Gonzalez AM, Paranhos R, Medeiros JA, Freitas FA, Albano RM, Martins OB (2009) Archaeal and bacterial communities facultatively autotrophic hydrogen oxidizing representatives of heavy metal contaminated acidic waters from zinc mine resi- of Hydrogenophaga can contribute in this process [44]. dues in Sepetiba bay. Extremophiles 13(2):263–271. https​://doi. 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