Microbial Ecology

, Volume 66, Issue 1, pp 5–18 | Cite as

Structure and Community Composition of Sprout-Like Bacterial Aggregates in a Dinaric Karst Subterranean Stream

  • Rok KostanjšekEmail author
  • Lejla Pašić
  • Holger Daims
  • Boris Sket
Microbiology of Aquatic Systems


The Vjetrenica cave in the Dinaric Karst hosts a worldwide extraordinarily high cave biodiversity. Beside a diverse and specialized cave fauna, sprout-like formations attached to the bed of the cave stream were observed and described, but not further characterized, almost a century ago. Here we investigated these sprout-like microbial aggregates by the rRNA approach and detailed microscopy. Based on fluorescence in situ hybridization and ultrastructural analysis, the sprout-like formations are morphologically highly organized, and their core consists of a member of a novel deep-branching lineage in the bacterial phylum Nitrospirae. This organism displays an interesting cellular ultrastructure with different kinds of cytoplasmic inclusions and is embedded in a thick extracellular matrix, which contributes to the stability and shape of the aggregates. This novel bacterium has been provisionally classified as “Candidatus Troglogloea absoloni.” The surface of the sprout-like aggregates is more diverse than the core. It is colonized by a bacterial biofilm consisting primarily of filamentous Betaproteobacteria, whereas other microbial populations present in the crust include members of the Bacteriodetes, Gammaproteobacteria, Actinombacteria, Alphaproteobacteria, and Planctomycetes, which are intermingled with mineral inclusions. This study represents the first thorough molecular and ultrastructural characterization of the elusive sprout-like bacterial aggregates, which are also found in other cave systems of the Dinaric Karst. The discovery of Ca. Troglogloea absoloni contributes to the known biodiversity of subterranean ecosystems and especially of macroscopic structures formed in caves by microorganisms, whose composition and ecological function often remain enigmatic.


Clone Library Betaproteobacteria Periplasmic Space Nitrite Oxidizer Karst Cave 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We are grateful to D. Bakšić for the photography of the sprout-like formations in their natural environment, J. Jugovic for the assistance in the sampling and measurements on field, B. Papić for the assistance in clone library preparation, R. Ozimec for providing scientific literature, and J. Dolinšek for the constructive suggestions on hybridization procedures. This work was financed by the Slovenian Research Agency (ARRS), research programs no. P1-0184 and P1-0198.

Supplementary material

248_2012_172_Fig9_ESM.jpg (41 kb)
Supplementary Fig. 1

ML phylogenetic tree showing the positions of phylotypes recovered from sprout-like microbial community clone libraries and belonging to a classes Gammaproteobacteria and Alphaproteobacteria of Proteobacteria and b Bacteroidetes, Actinobacteria, and Planctomycetes. Names in italics correspond to cultivated species, while the rest correspond to 16S rRNA gene clones. Names in bold correspond to the clones obtained in this study. The bullets indicate that the ML and MP bootstrap values and Bayesian posterior probabilities were ≥75 % (JPEG 41.1 kb)

248_2012_172_MOESM1_ESM.tif (2.3 mb)
High resolution image (TIFF 2.33 mb)
248_2012_172_MOESM2_ESM.docx (15 kb)
ESM 2 (DOCX 14.8 kb)


  1. 1.
    Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770PubMedGoogle Scholar
  2. 2.
    Amann RI, Ludwig W, Schleifer K-H (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169PubMedGoogle Scholar
  3. 3.
    Burdon KL (1946) Fatty material in bacteria and fungi revealed by staining dried, fixed slide preparation. J Bacteriol 52:665–678PubMedGoogle Scholar
  4. 4.
    Chen Y, Wu L, Boden R, Hillebrand A, Kumaresan D, Moussard H, Baciu M, Lu Y, Colin Murrell J (2009) Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave. ISME J 3:1093–1104PubMedCrossRefGoogle Scholar
  5. 5.
    Christiansen KA (1962) Proposition pour la classification des animaux cavernicoles. Spelunca 2:76–78Google Scholar
  6. 6.
    Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen ASD, McGarrell M, Marsh T, Garrity GM, Tiedje JM (2008) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucl Acids Res 37:D141–D145PubMedCrossRefGoogle Scholar
  7. 7.
    Crabtree K, McCoy E (1967) Zoogloea ramigera Itzigsohn, identification and description. Int J Syst Bacteriol 17:1–10CrossRefGoogle Scholar
  8. 8.
    Culver DC (1985) Trophic relationships in aquatic environments. Stygologia 1:43–53Google Scholar
  9. 9.
    Culver DC, Sket B (2000) Hotspots of subterranean biodiversity in caves and wells. J Cave Karst Stud 62:11–17Google Scholar
  10. 10.
    Daims H, Brühl A, Amann R, Schleifer K-H, Wagner M (1999) The domain-specific probe EUB338 is insufficient for the detection of all Bacteria; development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22:434–444PubMedCrossRefGoogle Scholar
  11. 11.
    Daims H, Nielsen JL, Nielsen PH, Scleifer K-H, Wagner M (2001) In situ characterization of Nitrospira-like nitrite-oxydizing bacteria in wastewater treatment plants. Appl Environ Microbiol 67:5273–5284PubMedCrossRefGoogle Scholar
  12. 12.
    Daims H, Stoecker K, Wagner M (2005) Fluorescence in situ hybridisation for the detection of prokaryotes. In: Osborn AM, Smith CJ (eds) Molecular microbial ecology. Bios-Garland, Abingdon, pp 213–239Google Scholar
  13. 13.
    DeLong EF (1992) Archaea in coastal marine environments. Proc Natl Acad Sci U S A 89:5685–5689PubMedCrossRefGoogle Scholar
  14. 14.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl Acids Res 32:1792–1797PubMedCrossRefGoogle Scholar
  15. 15.
    Ehrich S, Behrens D, Lebedeva E, Ludwig W, Bock E (1995) A new obligately chemolithoautotrophic, nitrite oxidizing bacterium, Nitrospira moscoviensis sp. nov., and its phylogenetic relationship. Arch Microbiol 164:16–23PubMedCrossRefGoogle Scholar
  16. 16.
    Engel AS (2010) Microbial diversity of cave ecosystems. In: Barton L, Mandl M, Loy A (eds) Geomicrobiology: Molecular, Environmental Perspectives. Springer, Berlin, pp 219–238CrossRefGoogle Scholar
  17. 17.
    Farnleitner A, Wilhartitz I, Kirschner AKT, Stadler H, Burtscher MM, Hornek R, Szewzyk U, Herndl G, Mach R (2005) Bacterial dynamics in spring water of alpine karst aquifers indicates the presence of stable autochthonous microbial endokarst communities. Environ Microbiol 7:1248–1259PubMedCrossRefGoogle Scholar
  18. 18.
    Friedman BA, Dugan PR, Pfister RM, Remsen CC (1968) Fine structure and composition of the Zoogloeal matrix surrounding Zoogloea ramigera. J Bacteriol 96:2144–2153PubMedGoogle Scholar
  19. 19.
    Friedman BA, Dugan PR (1968) Identification of Zoogloea species and the relationship to zoogloeal matrix and floc formation. J Bacteriol 95:1903–1909PubMedGoogle Scholar
  20. 20.
    Glöckner FO, Fuchs BM, Amman R (1999) Bacterioplankton compositions of lake and oceans: a first comparison based of fluorescence in situ hybridization. Appl Environ Microbiol 65:3721–3726PubMedGoogle Scholar
  21. 21.
    Good IJ (1953) The population frequencies of species and the estimation of population parameters. Biometrika 40:237–264Google Scholar
  22. 22.
    Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704PubMedCrossRefGoogle Scholar
  23. 23.
    Haouari O, Fardeau ML, Cayol JL, Fauque G, Casiot C, Elbaz-Poulichet F, Hamdi M, Ollivier B (2008) Thermodesulfovibrio hydrogeniphilus sp. nov., a new thermophilic sulphate-reducing bacterium isolated from a Tunisian hot spring. Syst Appl Microbiol 31:38–42PubMedCrossRefGoogle Scholar
  24. 24.
    Henry EA, Devereux R, Maki JS, Gilmour CC, Woese CR, Mandelco L, Schauder R, Remsen CC, Mitchell R (1994) Characterization of a new thermophilic sulfate-reducing bacterium Thermodesulfovibrio yellowstonii, gen. nov. and sp. nov.: its phylogenetic relationship to Thermodesulfobacterium commune and their origins deep within the bacterial domain. Arch Microbiol 161:62–69PubMedCrossRefGoogle Scholar
  25. 25.
    Hippe H (2000) Leptospirillum gen. nov. (ex Markosyan 1972), nom. rev., including Leptospirillum ferrooxidans sp. nov. (ex Markosyan 1972), nom. rev. and Leptospirillum thermoferrooxidans sp. nov. (Golovacheva et al. 1992). Int J Syst Evol Microbiol 50:501–503PubMedCrossRefGoogle Scholar
  26. 26.
    Holmes AJ, Tujula NA, Holley M, Contos A, James JM, Rogers P, Gillings MR (2001) Phylogenetic structure of unusual aquatic microbial formations in Nullarbor caves, Australia. Environ Microbiol 3:256–264PubMedCrossRefGoogle Scholar
  27. 27.
    Huang WE, Stoecker K, Griffiths R, Newbold L, Daims H, Whiteley AS, Wagner M (2007) Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function. Environ Microbiol 9:1878–1889PubMedCrossRefGoogle Scholar
  28. 28.
    Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2319PubMedCrossRefGoogle Scholar
  29. 29.
    Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian interface of phylogenetic trees. Bioinformatics 17:754–755PubMedCrossRefGoogle Scholar
  30. 30.
    Inglis TJJ, Sagripanti J-L (2006) Environmental factors that affect the survival and presistance of Burkholderia pseudomallei. Appl Environ Microbiol 72:6865–6875PubMedCrossRefGoogle Scholar
  31. 31.
    Jones DA, Lyon EH, Macalady JL (2009) Geomicrobiology of biovermiculations from the Frasassi cave system, Italy. J Cave Karst Stud 70:78–93Google Scholar
  32. 32.
    Jogler C, Niebler M, Lin W, Kube M, Wanner G, Kolinko S, Stief P, Beck AJ, De Beer D, Petersen N, Pan Y, Amann R, Reinhardt R, Schüler D (2010) Cultivation-independent characterization of 'Candidatus Magnetobacterium bavaricum' via ultrastructural, geochemical, ecological and metagenomic methods. Environ Microbiol 12(9):2466–2478PubMedCrossRefGoogle Scholar
  33. 33.
    Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematic. Wiley, New York, pp 115–175Google Scholar
  34. 34.
    Lebedeva EV, Off S, Zumbrägel S, Krause M, Shagzhina A, Lücker S, Maxiner F, Lipski A, Daims H, Spieck E (2011) Isolation and characterization of a moderately thermophilic nitrite-oxidizing bacterium from geithrmal spring. FEMS Microbiol Ecol 75:195–204PubMedCrossRefGoogle Scholar
  35. 35.
    Lechene CP, Luyten Y, McMahon G, Distel DL (2007) Quantitative imaging of nitrogen fixation by individual bacteria within animal cells. Science 317:1563–1566PubMedCrossRefGoogle Scholar
  36. 36.
    Lee N, Nielsen PH, Andreasen KH, Juretschko S, Nielsen JL, Schleifer K-H, Wagner M (1999) Combination of fluorescent in situ hybridization and microautoradiography—a new tool for structure-function analyses in microbial ecology. Appl Environ Microbiol 65:1289–1297PubMedGoogle Scholar
  37. 37.
    Loy A, Arnold R, Tischler P, Rattei T, Wagner M, Horn M (2008) probeCheck—a central resource for evaluating oligonucleotide probe coverage and specificity. Environ Microbiol 10:2894–2896PubMedCrossRefGoogle Scholar
  38. 38.
    Lučić I (2003) Vjetrenica, insight into the Earth's soul. ArTresor, ZagrebGoogle Scholar
  39. 39.
    Macalady JL, Lyon EH, Koffmann B, Albertson LK, Meyer K, Galdenzi S, Mariani S (2006) Dominant microbial populations in limestone-corroding stream biofilms, Frasassi cave system, Italy. Appl Environ Microbiol 72:5596–5609PubMedCrossRefGoogle Scholar
  40. 40.
    Macalady JL, Jones DS, Lyon EH (2007) Extremely acidic, pendulous cave wall biofilms from the Frasassi cave system, Italy. Environ Microbiol 9:1402–1414PubMedCrossRefGoogle Scholar
  41. 41.
    Manz W, Amann R, Ludwig W, Wagner M, Schleifer K-H (1992) Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst Appl Microbiol 15:593–600CrossRefGoogle Scholar
  42. 42.
    Markosyan GE (1972) A new iron-oxidizing bacterium—Leptospirillum ferrooxidans nov. gen. nov. sp. Biol J Armenia 25:26–29Google Scholar
  43. 43.
    Megušar F, Sket B (1977) On the nature of some organic covers on the cave walls. Proceedings of the 6th International Congress of Speleology. Olomouc 5:159–161Google Scholar
  44. 44.
    Murray RGE, Stackebrandt E (1995) Taxonomic note: implementation of the provisional status Candidatus for incompletely described procaryotes. Int J Syst Bacteriol 45:186–187PubMedCrossRefGoogle Scholar
  45. 45.
    Northup DE, Lavoie KH (2004) Microorganisms in caves. In: Gunn J (ed) Encyclopedia of caves and karst Sicene. Taylor and Francis Books, New York, pp 1083–1089Google Scholar
  46. 46.
    Northup DE, Melim LA, Spilde MN, Hathaway JMM, Garcia MG, Moya M, Stone FD, Boston PJ, Dapkevicius MLNE, Riquelme C (2011) Lava cave microbial communities within mats and secondary mineral deposits: implications for life detection on other planets. Astrobiology 11:601–618PubMedCrossRefGoogle Scholar
  47. 47.
    Ostle AG, Holt JG (1982) Nile blue A as a fluorescent stain for poly-3-hydroxybutyrate. Appl Environ Microbiol 44:238–241PubMedGoogle Scholar
  48. 48.
    Ozimec R, Lučić I (2009) The Vjetrenica cave (Bosnia and Herzegovina)—one of the world’s most prominent biodiversity hotspots for cave-dwelling fauna. Subterranean Biology 7:17–23Google Scholar
  49. 49.
    Pašić L, Kovče B, Sket B, Herzog-Velikonja B (2010) Diversity of microbial communities colonizing the walls of a Karstic cave in Slovenia. FEMS Microbiol Ecol 71:50–60PubMedCrossRefGoogle Scholar
  50. 50.
    Pleše B, Ozimec R, Tulić U, Ćetković H, Pojskić N, Lukić-Bilela L (2011) Unusual organic formations in kavernicole aquatic habitats of Dinarides. In: Ecosystems 2011: Structure and dynamics of ecosystems dinarides—status, possibilities and prospects—book of abstracts, Faculty of Science University of Sarajevo and Academy of Sciences and Arts of Bosnia and Herzegovina, Sarajevo, pp 65–66Google Scholar
  51. 51.
    Porca E, Jurado V, Žgur-Bertok D, Saiz-Jimenez C, Pašić L (2012) Comparative analysis of yellow microbial communities growing on the walls of geographically distinct caves indicates a common core of microorganisms involved in their formation. FEMS Microbiol Ecol 81:255–266PubMedCrossRefGoogle Scholar
  52. 52.
    Pronk M, Goldscheider N, Zopfi J (2009) Microbial communities in karst groundwater and their potential use for biomonitoring. Hydrogeol J 17:37–48CrossRefGoogle Scholar
  53. 53.
    Sarbu SM, Kane TC, Kinkle BK (1996) A chemoautotrophically based cave ecosystem. Science 272:1953–1955PubMedCrossRefGoogle Scholar
  54. 54.
    Schabereiter-Gurtner C, Saiz-Jimenez C, Piñar G, Lubitz W, Rölleke S (2002) Phylogenetic 16S rRNA analysis reveals the presence of complex and partly unknown bacterial communities in Tito Bustillo cave, Spain, and on its Palaeolithic paintings. Environ Microbiol 4:392–400PubMedCrossRefGoogle Scholar
  55. 55.
    Schabereiter-Gurtner C, Saiz-Jimenez C, Piñar G, Lubitz W, Rölleke S (2004) Phylogenetic diversity of bacteria associated with palaeolithic paintings and surrounding rock walls in two Spanish caves (Llonin and La Garma). FEMS Microbiol Ecol 47:235–247PubMedCrossRefGoogle Scholar
  56. 56.
    Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB et al (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541PubMedCrossRefGoogle Scholar
  57. 57.
    Sekiguchi Y, Muramatsu M, Imachi H, Narihiro T, Ohashi A, Harada H, Hanada S, Kamagata Y (2008) Thermodesulfovibrio aggregans sp. nov. and Thermodesulfovibrio thiophilus sp. nov., anaerobic, thermophilic, sulfate-reducing bacteria isolated from thermophilic methanogenic sludge, and emended description of the genus Thermodesulfovibrio. Int J Syst Evol Microbiol 58:2541–2548PubMedCrossRefGoogle Scholar
  58. 58.
    Sket B (1996) The ecology of the anchihaline caves. Trends in Ecology and Evolution 11:221–225PubMedCrossRefGoogle Scholar
  59. 59.
    Sket B (2003) Cave fauna, the particular case of Vjetrenica. In: Lučić I (ed) Vjetrenica, insight into the Earth's soul. ArTresor, Zagreb, pp 149–202Google Scholar
  60. 60.
    Sket B, Paragamian K, Trontelj P (2004) A census of the obligate subterranean fauna in the Balkan Peninsula. In: Griffiths HI, Krystufek B, Reed JM (eds) Balkan Biodiversity. Pattern and Process in Europe's Biodiversity Hotspot. Kluwer Academic Publishers, Dordrecht, pp 309–322Google Scholar
  61. 61.
    Sket B (2012) Diversity Patterns in the Dinaric Karst. In: White WB, Culver DC (eds) Encyclopedia of Caves. Academic Press, Chennai, pp 228–238CrossRefGoogle Scholar
  62. 62.
    Smolikova O (1919) Přispěvek k poznání temnostnich bakterií z jeskyň Balkánu. Časopis Moravskeho Musea Zemskeho 17–19:177–188Google Scholar
  63. 63.
    Sonne-Hansen J, Ahring BK (1999) Thermodesulfobacterium hveragerdense sp. nov., and Thermodesulfovibrio islandicus sp. nov., two thermophilic sulfate reducing bacteria isolated from a Icelandic hot spring. Syst Appl Microbiol 22:559–564PubMedCrossRefGoogle Scholar
  64. 64.
    Stahl DA, Amann R (1991) Development and application of nucleic acid probes in bacterial systematics. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematic. Wiley, New York, pp 205–248Google Scholar
  65. 65.
    Summers Engel A (2012) Chemoautotrophy. In: White WB, Culver DC (eds) Encyclopedia of Caves. Academic Press, Chennai, pp 125–134CrossRefGoogle Scholar
  66. 66.
    Summers Engel A (2012) Microbes. In: White WB, Culver DC (eds) Encyclopedia of Caves. Academic Press, Chennai, pp 490–499CrossRefGoogle Scholar
  67. 67.
    Swofford DL (2001) PAUP, Version 4.0b10 [computer software and manual]. Sinauer Associates, SunderlandGoogle Scholar
  68. 68.
    van Helvoort P-J, Griffioen J, Edmunds WM (2009) Occurrence and behavior of main inorganic pollutants in European groundwater. In: Quevauviller P, Foulliac A-M, Grath J, Ward R (eds) Groundwater Monitoring. Wiley, Chichester, pp 83–109Google Scholar
  69. 69.
    Wallner G, Amann R, Beisker W (1993) Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14:136–143PubMedCrossRefGoogle Scholar
  70. 70.
    Watson SW, Bock E, Valois FW, Waterbury JB, Schlosser U (1986) Nitrospira marina gen. nov., sp. nov, a chemolithotrophic nitrite-oxidizing bacterium. Arch Microbiol 144:1–7CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Rok Kostanjšek
    • 1
    Email author
  • Lejla Pašić
    • 1
  • Holger Daims
    • 2
  • Boris Sket
    • 1
  1. 1.Department of Biology, Biotechnical FacultyUniversity of LjubljanaLjubljanaSlovenia
  2. 2.Department of Microbial Ecology, Ecology CentreUniversity of ViennaViennaAustria

Personalised recommendations