Environmental Science and Pollution Research

, Volume 22, Issue 8, pp 5995–6003 | Cite as

Diversity of acidophilic prokaryotes at two acid mine drainage sites in Turkey

  • Pınar AytarEmail author
  • Catherine Melanie Kay
  • Mehmet Burçin Mutlu
  • Ahmet Çabuk
  • David Barrie Johnson
Research Article


The biodiversity of acidophilic prokaryotes in two acidic (pH 2.8–3.05) mine drainage (AMD) sites (Balya and Çan) in Turkey was examined using a combined cultivation-based and cultivation-independent approach. The latter included analyzing microbial diversity using fluorescent in situ hybridization (FISH), terminal restriction enzyme fragment length polymorphism (`T-RFLP), and quantitative PCR (qPCR). Numbers of cultivatable heterotrophic acidophilic bacteria were over an order of magnitude greater than those of chemolithotrophic acidophiles in both AMD ponds examined. Isolates identified as strains of Acidithiobacillus ferrivorans, Acidiphilium organovorum, and Ferrimicrobium acidiphilum were isolated from the Balya AMD pond, and others identified as strains of Leptospirillum ferriphilum, Acidicapsa ligni, and Acidiphilium rubrum from Çan AMD. Other isolates were too distantly related (from analysis of their 16S rRNA genes) to be identified at the species level. Archaeal diversity in the two ponds appeared to be far more limited. T-RFLP and qPCR confirmed the presence of Ferroplasma-like prokaryotes, but no archaea were isolated from the two sites. qPCR generated semiquantitative data for genera of some of the iron-oxidizing acidophiles isolated and/or detected, suggesting the order of abundance was Leptospirillum > Ferroplasma > Acidithiobacillus (Balya AMD) and Ferroplasma > Leptospirillum > Acidithiobacillus (Çan AMD).


Acidophile Acidic mine drainage Microbial diversity Turkey 



This study is based partly on the PhD thesis of P. Aytar. The study was supported by Eskisehir Osmangazi University Scientific Research Projects Committee (Project No.: 201119018).

Supplementary material

11356_2014_3789_MOESM1_ESM.docx (2.8 mb)
Supplementary Fig. 1 FISH analysis of Balya and Çan AMD using different oligonucleotide probes (DOCX 2824 kb)


  1. Akyol Z (2012) Balıkesir-Balya cevherli sahalarının jeolojisi, mineralojisi ve maden potansiyelinin değerlendirilmesi. İstanbul Yerbilimleri Dergisi 3:1–2Google Scholar
  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefGoogle Scholar
  3. Anton J, Llobet-Brossa E, Rodriguez-Valera F, Amann R (1999) Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds. Environ Microbiol 1:517–523CrossRefGoogle Scholar
  4. Baba A, Gurdal G, Sengunalp F, Ozay O (2008) Effects of leachant temperature and pH on leachability of metals from fly ash. A case study: Çan thermal power plant, province of Canakkale, Turkey. Environ Monit Assess 139:287–298CrossRefGoogle Scholar
  5. Baker BJ, Banfield JF (2003) Microbial communities in acid mine drainage. FEMS Microbiol Ecol 44:139–152CrossRefGoogle Scholar
  6. Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421CrossRefGoogle Scholar
  7. Blowes DW, Ptacek CJ, Jambor JL, Weisener CG, Paktunc, D, Gould, WD, Johnson DB (2013) The geochemistry of acid mine drainage. In: Treatise on Geochemistry, 2nd Edition, ElsevierGoogle Scholar
  8. Bond PL, Smriga SP, Banfield JF (2000a) Phylogeny of microorganisms populating a thick, subaerial, predominantly lithotrophic biofilm at an extreme acid mine drainage site. Appl Environ Microbiol 66:3842–3849CrossRefGoogle Scholar
  9. Bond PL, Druschel GK, Banfield JF (2000b) Comparison of acid mine drainage microbial communities in physically and geochemically distinct ecosystems. Appl. Environ Microbiol 66:4962–4971CrossRefGoogle Scholar
  10. Bowei C, Xingyu L, Wenyan L, Jiankang W (2009) Application of clone library analysis and real-time PCR for comparison of microbial communities in a low-grade copper sulfide ore bioheap leachate. J Ind Microbiol Biotechnol 36:1409–1416CrossRefGoogle Scholar
  11. Cole JR, Chai B, Marsh TL, Farris RJ, Wang Q, Kulam SA et al (2003) The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res 31:442–443CrossRefGoogle Scholar
  12. Coram NJ, Rawlings DE (2002) Molecular relationship between two groups of the genus Leptospirillum and the finding that Leptospirillum ferriphilum sp. nov. dominates South African commercial biooxidation tanks that operate at 40°C. Appl Environ Microbiol 68:838–845CrossRefGoogle Scholar
  13. Coupland K, Johnson DB (2008) Evidence that the potential for dissimilatory ferric iron reduction is widespread among acidophilic heterotrophic bacteria. FEMS Microbiol Lett 279:30–35CrossRefGoogle Scholar
  14. Dereeper A, Guignon V, Blanc G, Audic S, Buffet S et al (2008) robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 1:36 (Web Server Issue): W465–9.0Google Scholar
  15. Edwards KJ, Gihring TM, Banfield JF (1999) Seasonal variations in microbial populations and environmental conditions in an extreme acid mine environment. Appl Environ Microbiol 65:3627–3632Google Scholar
  16. Hallberg KB, Johnson DB (2007) Isolation, enumeration, growth, and preservation of acidophilic prokaryotes. In: Hurst CJ, Crawford RL, Garland JL, Lipson DA, Mills AL, Stetzenbach LD (eds) Manual of Environmental Microbiology, 3rd edn. ASM Press, Washington, DC, pp 1155–1165Google Scholar
  17. Hallberg KB, Gonzalez-Toril E, Johnson DB (2010) Acidithiobacillus ferrivorans, sp. nov.; facultatively anaerobic, psychrotolerant iron-, and sulfur-oxidizing acidophiles isolated from metal mine-impacted environments. Extremophiles 14:9–19CrossRefGoogle Scholar
  18. Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2319CrossRefGoogle Scholar
  19. Inaner H, Karayigit AI (2008) Concentration of major and trace elements in the Miocene lignite from the Çanakkale-Çan coalfiled, Turkey. Energ Source Part A 30:289–296CrossRefGoogle Scholar
  20. Johnson DB, Hallberg KB (2003) The microbiology of acidic mine waters. Res Microbiol 154:466–473CrossRefGoogle Scholar
  21. Johnson DB, Bacelar-Nicolau P, Okibe N, Thomas A, Hallberg KB (2009) Ferrimicrobium acidiphilum gen. nov., sp. nov. and Ferrithrix thermotolerans gen. nov., sp. nov.: heterotrophic, iron-oxidizing, extremely acidophilic Actinobacteria. Int J Syst Evol Microbiol 59:1082–1089CrossRefGoogle Scholar
  22. Johnson DB (2012) Geomicrobiology of extremely acidic subsurface environments. FEMS Microb Ecol 81:2–12CrossRefGoogle Scholar
  23. Kay CM, Rowe OF, Rocchetti L, Coupland K, Hallberg KB, Johnson DB (2013) Evolution of microbial “streamer” growths in an acidic metal-contaminated stream draining an abandoned underground copper mine. Life doi: 10.3390/life3010189#_blank 3:189-211
  24. Kimura S, Bryan CG, Hallberg KB, Johnson DB (2011) Biodiversity and geochemistry of an extremely acidic, low-temperature subterranean environment sustained by chemolithotrophy. Environ Microbiol 13:2092–2104CrossRefGoogle Scholar
  25. Kulichevskaya IS, Kostina LA, Valášková V, Rijpstra WIC, Damsté JSS, de Boer W, Svetlana N, Dedysh SN (2012) Acidicapsa borealis gen. nov., sp. nov. and Acidicapsa ligni sp. nov., subdivision 1 Acidobacteria from Sphagnum peat and decaying wood. Int J Syst Evol Microbiol 62:1512–1520CrossRefGoogle Scholar
  26. Liljeqvist M, Valdes J, Holmes DS, Dopson M (2011) Draft genome of the psychrotolerant acidophile Acidithiobacillus ferrivorans SS3. J Bacteriol 193:4304–4305CrossRefGoogle Scholar
  27. Lobos JH, Chisolm TE, Bopp LH, Holmes DS (1986) Acidiphilium organovorum sp. nov., an acidophilic heterotroph isolated from a Thiobacillus ferrooxidans culture. Int J. Syst Bacteriol 36:139–144CrossRefGoogle Scholar
  28. Lopez-Archilla AI, Marín I, Amils R (2001) Microbial community composition and ecology of an acidic aquatic environment: the Tinto River, Spain. Microbial Ecol 41:20–35Google Scholar
  29. Snaidr MO, Amann R, Huber I, Ludwig W, Schleifer KH (1997) Phylogenetic analysis and in situ identification of bacteria in activated sludge. Appl Environ Microbiol 63:2884–2896Google Scholar
  30. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefGoogle Scholar
  31. Vera M, Schippers A, Sand W (2013) Progress in bioleaching: fundamentals and mechanism of bacterial metal sulfide oxidation-part A. Appl Microbiol Biotechnol 97:7529–7541CrossRefGoogle Scholar
  32. Wichlacz PL, Unz RF, Langworthy TA (1986) Acidiphilium angustum sp. nov., Acidiphilium facilis sp. nov., and Acidiphilium rubrum sp. nov.: acidophilic heterotrophic bacteria isolated from acicid coal mine drainage. Int J Syst Bacteriol 36:197–201CrossRefGoogle Scholar
  33. Zammit CM, Mutch LA, Watling HR, Watkin ELJ (2008) Evaluation of quantitative real-time polymerase chain reaction for enumeration of biomining microorganisms in culture. Hydrometallurgy 94:185–189CrossRefGoogle Scholar
  34. Zhang R, Wei M, Ji H, Chen X, Qiu G, Zhou H (2009) Application of real-time PCR to monitor population dynamics of defined mixed cultures of moderate thermophiles involved in bioleaching of chalcopyrite. Appl Microbiol Biotechnol 81:1161–1168CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Pınar Aytar
    • 1
    Email author
  • Catherine Melanie Kay
    • 3
  • Mehmet Burçin Mutlu
    • 2
  • Ahmet Çabuk
    • 1
  • David Barrie Johnson
    • 3
  1. 1.Department of Biotechnology and Biosafety, Graduate School of Natural and Applied SciencesEskisehir Osmangazi UniversityEskisehirTurkey
  2. 2.Department of Biology, Faculty of ScienceAnadolu UniversityEskisehirTurkey
  3. 3.College of Natural SciencesBangor UniversityBangorUK

Personalised recommendations