, Volume 23, Issue 1, pp 35–48 | Cite as

High diversity of thermophilic cyanobacteria in Rupite hot spring identified by microscopy, cultivation, single-cell PCR and amplicon sequencing

  • Otakar StruneckýEmail author
  • Karel Kopejtka
  • Franz Goecke
  • Jürgen Tomasch
  • Jaromír Lukavský
  • Amir Neori
  • Silke Kahl
  • Dietmar H. Pieper
  • Plamen Pilarski
  • David Kaftan
  • Michal Koblížek
Original Paper


Genotypic and morphological diversity of cyanobacteria in the Rupite hot spring (Bulgaria) was investigated by means of optical microscopy, cultivation, single-cell PCR, and 16S rRNA gene amplicon sequencing. Altogether, 34 sites were investigated along the 71–39 °C temperature gradient. Analysis of samples from eight representative sites shown that Illumina, optical microscopy, and Roche 454 identified 72, 45 and 19% respective occurrences of all cumulatively present taxa. Optical microscopy failed to detect species of minor occurrence; whereas, amplicon sequencing technologies suffered from failed primer annealing and the presence of species with extensive extracellular polysaccharides production. Amplicon sequencing of the 16S rRNA gene V5–V6 region performed by Illumina identified the cyanobacteria most reliably to the generic level. Nevertheless, only the combined use of optical microscopy, cultivation and sequencing methods allowed for reliable estimate of the cyanobacterial diversity. Here, we show that Rupite hot-spring system hosts one of the richest cyanobacterial flora reported from a single site above 50 °C. Chlorogloeopsis sp. was the most abundant at the highest temperature (68 °C), followed by Leptolyngbya boryana, Thermoleptolyngbya albertanoae, Synechococcus bigranulatus, Oculatella sp., and Desertifilum sp. thriving above 60 °C, while Leptolyngbya geysericola, Geitlerinema splendidum, and Cyanobacterium aponinum were found above 50 °C.


Bulgaria Cyanobacteria Extremophile Hot spring 



We thank M. Dachev, H. Medová, V. Selyanin, T. Stambolieva and V. Titlová for their technical assistance. The authors are particularly indebted to the editor and reviewers for their valuable comments on the manuscript. This study was conducted with support from GAČR projects 15-00703S (to M.K., D.K. and K.K.), and 15-00113S (to O.S.), Czech Ministry of Education projects “CENAKVA” (No. CZ.1.05/2.1.00/01.0024), and “CENAKVA II” (No. LO1205 under the NPU I program), European Regional Development Fund-Project (No. CZ.02.1.01/0.0/0.0/15_003/0000441) and Algatech Plus (LO1416). J. L. and P.P. thank the Mobility Program of the Bulgarian and Czech Academies of Science, “Study of biotechnological potential of extremophilic and extremotolerant algae and cyanobacteria”.

Author contributions

MK designed the study. OS, FG, JL, PP, DK conducted the collections and strain isolations. OS, KK, JT, SK, and DHP conducted sequencing, sequence analyses and bioinformatics. OS, MK, FG, DK and AN wrote the paper.

Compliance with ethical standards

Conflict of interest

No potential conflict of interests was reported by the authors.

Supplementary material

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Supplementary material 1 (PDF 1651 kb)
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Supplementary material 2 (PDF 422 kb)
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Supplementary material 3 (PDF 97 kb)
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Supplementary material 4 (PDF 190 kb)
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Supplementary material 5 (PDF 168 kb)


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Copyright information

© Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Otakar Strunecký
    • 1
    • 9
    Email author
  • Karel Kopejtka
    • 2
  • Franz Goecke
    • 2
    • 4
  • Jürgen Tomasch
    • 5
  • Jaromír Lukavský
    • 3
  • Amir Neori
    • 6
  • Silke Kahl
    • 7
  • Dietmar H. Pieper
    • 7
  • Plamen Pilarski
    • 8
  • David Kaftan
    • 2
    • 9
  • Michal Koblížek
    • 2
  1. 1.Institute of Aquaculture, CENAKVA, Faculty of Fisheries and Protection of WatersUniversity of South BohemiaČeské BudějoviceCzech Republic
  2. 2.Center AlgatechInstitute of Microbiology CASTřeboňCzech Republic
  3. 3.Department of Plant EcologyInstitute of Botany CASTřeboňCzech Republic
  4. 4.Department of Plant and Environmental ScienceNorwegian University of Life SciencesÅsNorway
  5. 5.Group Microbial CommunicationHelmholtz Centre for Infection ResearchBrunswickGermany
  6. 6.Israel Oceanographic and Limnological Research Ltd., National Center for MaricultureEilatIsrael
  7. 7.Group Microbial Interactions and ProcessesHelmholtz Centre for Infection ResearchBrunswickGermany
  8. 8.Institute of Plant Physiology and Genetics BASSofiaBulgaria
  9. 9.Faculty of ScienceUniversity of South BohemiaČeské BudějoviceCzech Republic

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