, Volume 70, Issue 8, pp 989–1002 | Cite as

Down into the Earth: microbial diversity of the deepest cave of the world

  • Ieva Kieraite-Aleksandrova
  • Vilius Aleksandrovas
  • Nomeda KuisieneEmail author


In our work, microbial diversity of Krubera-Voronja cave was evaluated in the view of the frequency of human visits in different locations as well as the sampling depth. Sampling in this cave was performed at depths of 220 m to 1640 m. Cultivation-independent method, namely barcoded pyrosequencing of 16S rRNA gene, was used for this analysis. Our results demonstrated high bacterial diversity at the phylum and genus levels. We have shown that the bacterial diversity at the phylum level depends on both the sampling depth and the frequency of human visits in Krubera-Voronja cave. Frequently visited locations were more diverse at the phylum level than the rarely visited branches. The total number of bacterial genera both per phylum and per sample correlated with the frequency of human visits but not with the sampling depth. Some genera, found in Krubera-Voronja cave, seem to be absent or very rare in other caves. The present study represents the first report on the microbial diversity in Krubera-Voronja cave.

Key words

pyrosequencing Krubera-Voronja speleomicrobiology 16S rRNA gene deep cave microbial diversity 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



This work was supported by the Research Council of Lithuania (grant No. MIP-005/2014) and the National Geographic’s Global Exploration Fund (grant No. GEFNE50-Y12). Lithuanian caving club ‘Aenigma’ and Ukrainian Speleological Association are acknowledged for their cooperation in sampling. We would like to express our gratitude to Vytautas Zurauskas for his valuable consultations and assistance with bioinformatics.


  1. Barton H.A., Taylor M.R. & Pace N.R. 2004. Molecular phylogenetic analysis of a bacterial community in an oligotrophic cave environment. Geomicrobiol. J. 21: 11–20.CrossRefGoogle Scholar
  2. Bastian R., Alabouvette C. & Saiz-Jimenez C. 2009. Bacteria and free-living amoeba in the Lascaux cave. Res. Microbiol. 160: 38–40.CrossRefGoogle Scholar
  3. Carmichael M.J., Carmichael S.K., Santelli C.M., Strom A. & Bräuer S.L. 2013. Mn(II)-oxidizing bacteria are abundant and environmentally relevant members of ferromanganese deposits in caves of the Upper Tennessee River Basin. Geomicrobiol. J. 30: 779–800.CrossRefGoogle Scholar
  4. Chakravorty S., Helb D., Burday M., Connell N. & Alland D. 2007. A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. J. Microbiol. Methods 69: 330–339.CrossRefGoogle Scholar
  5. Chan K.G. & Chong T.M. 2014. Prevalence of unclassified bacteria in tropical coastal waters of Malaysia revealed by metagenomic approach. Genome Announc. 2: e00419-14.Google Scholar
  6. Clarke K.R. 1993. Non-parametric multivariate analyses of changes in community structure. Aust. J. Ecol. 18: 117–143.CrossRefGoogle Scholar
  7. Cole J.R., Wang Q., Fish J.A., Chai B., McGarrell D.M., Sun Y., Brown C.T., Porras-Alfaro A., Kuske C.R. & Tiedje J.M. 2014. Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res. 42: D633–D642.CrossRefGoogle Scholar
  8. Cuezva S., Fernandez-Cortes A., Porca E., Pašić L., Jurado V., Hernandez-Marine M., Serrano-Ortiz P., Hermosin B., Cañaveras J.C., Sanchez-Moral S. & Saiz-Jimenez C. 2012. The biogeochemical role of Actinobacteria in Altamira Cave, Spain. FEMS Microbiol. Ecol. 81: 281–290.CrossRefGoogle Scholar
  9. Dillies M.A., Rau A., Aubert J., Hennequet-Antier C., Jeanmougin M., Servant N., Keime C., Marot G., Castel D., Estelle J., Guernec G., Jagla B., Jouneau L., Laloë D., Le Gall C., Schaëffer B., Le Crom S., Guedj M. & Jaffrézic F. 2013. A comprehensive evaluation of normalization methods for Illumina high-throughput RNA sequencing data analysis. Brief. Bioinformatics 14: 671–683.CrossRefGoogle Scholar
  10. Edgar R.C., Haas B.J., Clemente J.C., Quince C. & Knight R. 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27: 2194–2200.CrossRefGoogle Scholar
  11. Engei A.S. 2010. Microbial diversity of cave ecosystems, pp. 219–238. In: Loy A., Mandl M. & Barton L. (eds), Geomicro-biology: Molecular and Environmental Perspective, Springer Science+Business Media B.V., Dordrecht.Google Scholar
  12. Engel A.S., Paoletti M.G., Beggio M., Dorigo L., Pamio A., Gomiero T., Furlan C., Brilli M., Dreon A.L., Bertoni R. & Squartini A. 2013. Comparative microbial community composition from secondary carbonate (moonmilk) deposits: implications for the Cansiliella servadeii cave hygropetric food web. Int. J. Speleol. 42: 181–192.CrossRefGoogle Scholar
  13. Epure L., Meleg I.N., Munteanu C.-M., Roban R.D. & Moldovan O.T. 2014. Bacterial and fungal diversity of quaternary cave sediment deposits. Geomicrobiol. J. 31: 116–127.CrossRefGoogle Scholar
  14. Gan H.Y., Gan H.M., Tarasco A.M., Busairi N.I., Barton H.A., Hudson A.O. & Savka M.A. 2014. Whole-genome sequences of five oligotrophic bacteria isolated from deep within Lechuguilla Cave, New Mexico. Genome Announc. 2: e01133-14.Google Scholar
  15. Griffin D.W., Gray M.A., Lyles M.B. & Northup D.E. 2014. The transport of nonindigenous microorganisms into caves by human visitation: a case study at Carlsbad Caverns National Park. Geomicrobiol. J. 31: 175–185.CrossRefGoogle Scholar
  16. Groth I., Schumann P., Laiz L., Sanchez-Moral S., Cahveras J.C. & Saiz-Jimenez C. 2001. Geomicrobiological study of the Grotta dei Cervi, Porto Badisco, Italy. Geomicrobiol. J. 18: 241–258.CrossRefGoogle Scholar
  17. Jones D.S., Schaperdoth I. & Macalady J.L. 2014. Metage-nomic evidence for sulfide oxidation in extremely acidic cave biofilms. Geomicrobiol. J. 31: 194–204.CrossRefGoogle Scholar
  18. Jurado V., Laiz L., Rodriguez-Nava V., Boiron P., Boiron P., Hermosin B., Sanchez-Moral S. & Saiz-Jimenez C. 2010. Pathogenic and opportunistic microorganisms in caves. Int. J. Speleol. 39: 15–24.CrossRefGoogle Scholar
  19. Klindworth A., Pruesse E., Schweer T., Peplies J., Quast C., Horn M. & Glockner F.O. 2013. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 41: e1.Google Scholar
  20. Kodama Y., Shumway M. & Leinonen R. 2012. The sequence read archive: explosive growth of sequencing data. Nucleic Acids Res. 40 (Database Issue): D54–D56.CrossRefGoogle Scholar
  21. Kumaresan D., Wischer D., Stephenson J., Hillebrand-Voiculescu A. & Murrell J.C. 2014. Microbiology of Movile Cave a chemolithoautotrophic ecosystem. Geomicrobiol. J. 31: 186–193.CrossRefGoogle Scholar
  22. McLellan S.L. & Eren A.M. 2014. Discovering new indicators of fecal pollution. Trends Microbiol. 22: 697–706.CrossRefGoogle Scholar
  23. Northup D.E. & Lavoie K.H. 2001. Geomicrobiology of caves: a review. Geomicrobiol. J. 18: 199–222.CrossRefGoogle Scholar
  24. Northup D.E., Melim L.A., Spilde M.N., Hathaway J.J.M., Garcia M.G., Moya M., Stone F.D., Boston P.J., Dapkevicius M.L.N.E. & Riquelme C. 2011. Lava cave microbial communities within mats and secondary mineral deposits: implications for life detection on other planets. Astrobiology 11: 601–618.CrossRefGoogle Scholar
  25. Ortiz M., Neilson J.W., Nelson W.M., Legatzki A., Byrne A., Yu Y., Wing R.A., Soderlund C.A., Pryor B.M., Pierson III L.S. & Maier R.M. 2013. Profiling bacterial diversity and taxonomic composition on speleothem surfaces in Kartchner Caverns, AZ. Microb. Ecol. 65: 371–383.CrossRefGoogle Scholar
  26. Porat L., Vishnivetskaya T.A., Mosher J.J., Brandt C.C., Yang Z.K., Brooks S.C., Liang L., Drake M.M., Podar M., Brown S.D., Palumbo A.V. 2010. Characterization of archaeal community in contaminated and uncontaminated surface stream sediments. Microb. Ecol. 60: 784–795.CrossRefGoogle Scholar
  27. Porter M.L., Engel A.S., Kane T.C. & Kinkle B.K. 2009. Productivity-diversity relationships from chemolithoautotrophically based sulfidic karst systems. Int. J. Speleol. 38: 27–40.CrossRefGoogle Scholar
  28. Portillo M.C., Gonzalez J.M. & Saiz-Jimenez C. 2008. Metabolically active microbial communities of yellow and grey colonizations on the walls of Altamira Cave, Spain. J. Appl. Microbiol. 104: 681–691.CrossRefGoogle Scholar
  29. Portillo M.C., Saiz-Jimenez C. & Gonzalez J.M. 2009. Molecular characterization of total and metabolically active bacterial communities of “white colonizations’ in the Altamira Cave, Spain. Res. Microbiol. 160: 41–47.CrossRefGoogle Scholar
  30. Schabereiter-Gurtner C., Saiz-Jimenez C., Pińar G., Lubitz W. & Rolleke S. 2002. Altamira cave Paleolithic paintings harbor partly unknown bacterial communities. FEMS Microbiol. Lett. 211: 7–11.CrossRefGoogle Scholar
  31. Sendra A. & Reboleira A.S.P.S. 2012. The world’s deepest subterranean community Krubera-Voronja Cave (Western Caucasus). Int. J. Speleol. 41: 221–230.CrossRefGoogle Scholar
  32. Studholme D.J., Jackson R.A. & Leak D.J. 1999. Phylogenetic analysis of transformable strains of thermophilic Bacillus species. FEMS Microbiol. Lett. 172: 85–90.CrossRefGoogle Scholar

Copyright information

© Slovak Academy of Sciences 2015

Authors and Affiliations

  • Ieva Kieraite-Aleksandrova
    • 1
  • Vilius Aleksandrovas
    • 1
  • Nomeda Kuisiene
    • 1
    Email author
  1. 1.Department of Microbiology and BiotechnologyVilnius UniversityVilniusLithuania

Personalised recommendations