RESEARCH ARTICLE Diversity of acidophilic prokaryotes at two acid mine drainage sites in Turkey Pınar Aytar & Catherine Melanie Kay & Mehmet Burçin Mutlu & Ahmet Çabuk & David Barrie Johnson Received: 15 July 2014 /Accepted: 28 October 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract 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 includ- ed analyzing microbial diversity using fluorescent in situ hy- bridization (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). Keywords Acidophile . Acidic mine drainage . Microbial diversity . Turkey Introduction Acid mine drainage (AMD), which is frequently characterized by low pH values (often <3), and elevated concentrations of sulfate, iron, and other metals and metalloids forms when sulfide minerals such as pyrite (FeS 2 ) are exposed to both air and water (Blowes et al. 2013). The oxidative dissolution of these minerals can be greatly accelerated by some acidophilic iron- and sulfur-oxidizing bacteria and archaea, such as Acidithiobacillus, Leptospirillum, and Ferroplasma spp. (John- son 2012). Prokaryotes that catalyze the dissimilatory oxidation of ferrous iron are considered to be the primary microbial agents involved in degrading sulfides as the ferric iron produced is the main oxidant of these minerals in acidic liquors (Vera et al. 2013). Oxidation of the reduced sulfur compounds that form during the oxidation of sulfide minerals by sulfur-oxidizing acidophilic prokaryotes generates sulfuric acid, which causes the acidification of impacted water bodies (streams, lakes, etc.; Liljeqvist et al. 2011). While the most frequently reported iron- and sulfur-oxidizing acidophiles in AMD are obligately auto- trophic (e.g., Acidithiobacillus and Leptospirillum spp.), others are facultative (e.g., Sulfobacillus spp.) or obligate (e.g., Ferrimicrobium acidiphilum) heterotrophs (Johnson et al. 2009). Some other species of obligately heterotrophic acido- philes that thrive in AMD (e.g., most Acidiphilium and Acidocella spp.) do not oxidize ferrous iron, though they can Responsible editor: Zhihong Xu Electronic supplementary material The online version of this article (doi:10.1007/s11356-014-3789-4) contains supplementary material, which is available to authorized users. P. Aytar (*) : A. Çabuk Department of Biotechnology and Biosafety, Graduate School of Natural and Applied Sciences, Eskisehir Osmangazi University, 26480 Eskisehir, Turkey e-mail: pinaraytar@gmail.com M. B. Mutlu Department of Biology, Faculty of Science, Anadolu University, Eskisehir, Turkey C. M. Kay : D. B. Johnson College of Natural Sciences, Bangor University, Deiniol Road, Bangor LL57 2UW, UK Environ Sci Pollut Res DOI 10.1007/s11356-014-3789-4